In situ expression of lipase for enzymatic production of alcohol esters during fermentation

ABSTRACT

Disclosed herein are methods of producing alcohol esters during a fermentation by providing alcohol-producing microorganisms which further comprise an engineered polynucleotide encoding a polypeptide having lipase activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of and claims the benefit ofpriority of U.S. application Ser. No. 13/427,987 filed on Mar. 23, 2012,which claims the benefit of priority of U.S. Provisional PatentApplication No. 61/466,712, filed Mar. 23, 2011 and U.S. ProvisionalPatent Application No. 61/498,292, filed Jun. 17, 2011. The contents ofthe referenced applications are herein incorporated by reference intheir entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: 20120615_CL5145USCIP_SeqList_ST25.txt, Size: 637,685bytes, and Date of Creation: Jun. 14, 2012) filed with the applicationis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the fermentative production ofalcohols, including ethanol and butanol, and processes for improvingalcohol fermentation employing in situ product removal methods.

BACKGROUND OF THE INVENTION

Alcohols have a variety of applications in industry and science. Forexample, alcohols can be used as a beverage (i.e., ethanol), fuel,reagents, solvents, and antiseptics. For example, butanol is an alcoholthat is an important industrial chemical with a variety of applications,including use as a fuel additive, as a feedstock chemical in theplastics industry, and as a food-grade extractant in the food and flavorindustry. Accordingly, there is a high demand for alcohols, such asbutanol, as well as for efficient production methods which do not relyon non-renewable resources.

Production of alcohol utilizing fermentation by microorganisms is onesuch production method which utilizes substrates from renewablefeedstocks. In the production of butanol in particular, somemicroorganisms that produce butanol in high yields also have low butanoltoxicity thresholds, such that butanol needs to be removed from thefermentation vessel as it is being produced. Thus, there is a continuingneed to develop efficient methods and systems for producing butanol inhigh yields despite low butanol toxicity thresholds of thebutanol-producing microorganisms in the fermentation medium. In situproduct removal (ISPR) can be used to remove butanol (or otherfermentative alcohol) from the fermentation vessel as it is produced,thereby allowing the microorganism to produce butanol at high yields.One method for ISPR for removing fermentative alcohol that has beendescribed in the art is liquid-liquid extraction (U.S. Patent Appl. Pub.No. 20090305370). In general, with regard to butanol fermentation, forexample, the fermentation medium is contacted with an organicextractant. The organic extractant and the fermentation medium form abiphasic mixture. The butanol partitions into the organic extractantphase, decreasing the concentration in the aqueous phase which contactsthe microorganism, thereby limiting the exposure of the microorganism tothe inhibitory butanol. Liquid-liquid extraction results from contactbetween the extractant and the fermentation broth for transfer of theproduct alcohol into the extractant; separation of the extractant phasefrom the aqueous phase; and, preferably, recycle of the extractant withminimal degradation of the partition coefficient of the extractant overa long-term operation.

The extractant can become contaminated over time with each recycle by,for example, the build-up of lipids present in the biomass that is fedto the fermentation vessel as feedstock of hydrolysable starch. As anexample, a liquified corn mash loaded to a fermentation vessel canresult in a fermentation broth that contains corn oil during conversionof glucose to butanol by simultaneous saccharification and fermentation(with saccharification of the liquified mash occurring duringfermentation by the addition of glucoamylase to produce glucose). Thedissolution of the corn oil lipids into an extractant during ISPR canresult in build-up of lipid concentration with each extractant recycle,decreasing the partition coefficient for the product alcohol inextractant as the lipid concentration in extractant increases with eachrecycle.

Converting the lipids present in a liquefied mash into an extractantthat can be used in ISPR is a method of decreasing the amount of lipidsthat are fed to the fermentation vessel, as is esterifying the productalcohol as it is produced during the fermentation with a fatty acid byadding lipase as an esterification catalyst to the fermentation. Suchmethods are described for example in US Appl. Pub. Nos. 20110312044 and20110312043, and PCT Appl. Pub. No. WO2011/159998, which are hereinincorporated by reference.

There is a continuing need for alternative extractive fermentationmethods which can also reduce costs associated with adding lipase to thefermentation.

SUMMARY OF THE INVENTION

Provided herein are methods comprising: a) providing a fermentationmedium comprising fermentable carbon substrate derived from a biomassfeedstock, alcohol produced from a fermentable carbon substrate derivedfrom a biomass feedstock, and an alcohol producing microorganism whereinthe alcohol producing microorganism comprises a polynucleotide encodinga polypeptide having lipase activity and the microorganism expresses anddisplays or secretes said polypeptide such that the lipase activity ispresent in the fermentation medium; b) contacting the fermentationmedium with a carboxylic acid; wherein the lipase activity is present inthe fermentation medium in sufficient amount to convert at least aportion of the alcohol produced by the microorganism to alcohol estersextracellularly. In embodiments, the alcohol producing microorganism isyeast. In embodiments, the polynucleotide encoding a polypeptide havinglipase activity is engineered. In embodiments, the methods furthercomprise contacting the fermentation medium with an extractant to form atwo-phase mixture comprising an aqueous phase and an organic phase. Inembodiments, the extractant comprises the carboxylic acid. Inembodiments, the product alcohol is a C₂ to C₈ alkyl alcohol. Inembodiments, the product alcohol is ethanol. In embodiments, the alcoholesters comprise fatty acid ethyl esters. In embodiments, the productalcohol is butanol. In embodiments, the alcohol esters comprise fattyacid butyl esters. In embodiments, the alcohol esters further comprisefatty acid ethyl esters.

In embodiments, polypeptides provided herein having lipase activity aredisplayed on the surface of the microorganism. In embodiments,polypeptides having lipase activity are secreted. In embodiments, thepolypeptide having lipase activity comprises a sequence having at leastabout 70% identity, at least about 80% identity, at least about 90%identity, or at least about 95% identity to any one of SEQ ID NOs: 249,250, 251, 252, 253 or a fragment thereof. In embodiments, thepolynucleotide encoding a polypeptide having lipase activity comprises asequence with at least about 70% identity to a polynucleotide having SEQID NO: 1, 3, 5, 7, 8, 9, 46, 48, 50, 52, 54, 255, 271 or 273. Inembodiments, the polypeptide having lipase activity comprises a sequencewith at least about 70% identity, at least about 80% identity, at leastabout 90% identity, or at least about 95% identity to a polypeptidehaving SEQ ID NO: 2, 4, 6, 256, 47, 49, 51, 53, 55, 241, 242, 243, 244,245, 246, 247, 248, 272, or 274 or an active fragment thereof. Inembodiments, the polypeptide having lipase activity does not contain aglycosylation motif. In embodiments, the polypeptide having lipaseactivity is not glycosylated.

In embodiments, the carboxylic acid comprises free fatty acids derivedfrom corn oil, canola oil, palm oil, linseed oil, jatropha oil, orsoybean oil. In embodiments, the carboxylic acid is derived from thesame biomass feedstock as the fermentable carbon substrate. Inembodiments, the carboxylic acid comprises carboxylic acids having C₁₂to C₂₂ linear or branched aliphatic chains. In embodiments, thecontacting with extractant and the contacting with carboxylic acid occurcontemporaneously. In embodiments, at least about 60% of the effectivetiter of alcohol produced by the microorganism is converted to alcoholesters. In embodiments, the fermentation medium further comprisestriglycerides, diglycerides, monoglycerides, and phospholipids, orcombinations thereof and the lipase activity hydrolyzes at least aportion of the triglycerides, diglycerides, monoglycerides, andphospholipids, or combinations thereof to form free fatty acids.

In embodiments, the effective titer of alcohol produced during afermentation is greater than that produced during a fermentation by analcohol-producing microorganism that does not comprise a polynucleotideencoding a polypeptide having lipase activity and the microorganismexpresses and secretes or displays said polypeptide such that the lipaseactivity is present in the fermentation medium. In embodiments, theeffective rate of alcohol produced during a fermentation is greater thanthe rate of alcohol production during a fermentation by analcohol-producing microorganism that does not comprise a polynucleotideencoding a polypeptide having lipase activity and the microorganismexpresses and secretes or displays said polypeptide such that the lipaseactivity is present in the fermentation medium.

Also provided herein are recombinant host cells comprising an engineeredalcohol production pathway; and an engineered polynucleotide encoding apolypeptide having lipase activity. In embodiments, the polypeptidehaving lipase activity comprises a sequence having at least about 70%identity, at least about 80% identity, at least about 90% identity, orat least about 95% identity to SEQ ID NO: 2, 4, 6, 256, 47, 49, 51, 53,55, 241, 242, 243, 244, 245, 246, 247, 248, 272, or 274 or an activefragment thereof. In embodiments, the polypeptide having lipase activitycomprises a sequence having at least about 70% identity, at least about80% identity, at least about 90% identity, or at least about 95%identity to any one of SEQ ID NOs: 249, 250, 251, 252, 253 or a fragmentthereof. In embodiments, the polypeptide having lipase activity does notcontain a glycosylation motif. In embodiments, the polypeptide havinglipase activity is not glycosylated. In embodiments, the engineeredpolynucleotide encoding a polypeptide having lipase activity comprises asequence having at least about 70% identity, at least about 80%identity, at least about 90% identity, or at least about 95% identity toSEQ ID NO: 1, 3, 5, 7, 8, 9, 46, 48, 50, 52, 54, 255, 271 or 273.

Also provided herein are recombinant host cells comprising an alcoholproduction pathway; and an engineered polynucleotide encoding apolypeptide having lipase activity wherein the polypeptide having lipaseactivity comprises a sequence having at least about 70% identity, atleast about 80% identity, at least about 90% identity, or at least about95% identity to SEQ ID NO: 2, 4, 6, 256, 47, 49, 51, 53, 55, 241, 242,243, 244, 245, 246, 247, 248, 272, or 274 or an active fragment thereof.In embodiments, the polypeptide having lipase activity further comprisesa sequence having at least about 70% identity, at least about 80%identity, at least about 90% identity, or at least about 95% identity toany one of SEQ ID NOs: 249, 250, 251, 252, 253 or a fragment thereof. Inembodiments, the alcohol production pathway is a butanol productionpathway. In embodiments, the butanol production pathway is an isobutanolproduction pathway. In embodiments, the host cell further comprisesreduced or eliminated pyruvate decarboxylase activity.

Also provided herein are methods of increasing tolerance of analcohol-producing microorganism to the produced alcohol, the methodscomprising: engineering a microorganism to express and secrete ordisplay a polypeptide having lipase activity; contacting the engineeredmicroorganism with triglycerides, diglycerides, monoglycerides,phospholipids, free fatty acids, or a mixture thereof and a carbonsubstrate under conditions whereby the microorganism produces analcohol. In embodiments, the engineered microorganism is contacted withtriglycerides, diglycerides, monoglycerides, and phospholipids, orcombinations thereof and wherein the secreted or displayed lipaseconverts at least a portion of the trigylcerides, diglycerides,monoglycerides, and phospholipids, or combinations thereof into freefatty acids. In embodiments, the lipase catalyzes the formation ofalcohol esters. In embodiments, the microorganism produces alcohol at aneffective titer greater than that produced by a microorganism that hasnot been engineered to express and secrete a polypeptide with lipaseactivity. In embodiments, the microorganism further comprises anengineered alcohol biosynthetic pathway. In embodiments, the engineeredalcohol biosynthetic pathway is a 1-butanol, a 2-butanol, or anisobutanol biosynthetic pathway. In embodiments, the isobutanolbiosynthetic pathway comprises the following substrate to productconversions: pyruvate to acetolactate, acetolactate to2,3-dihydroxyisovalerate, 2,3-dihydroxyisovalerate to 2-ketoisovalerate,2-ketoisovalerate to isobutyraldehyde; and, isobutyraldehyde toisobutanol.

Provided herein are methods of producing butyl esters during afermentation comprising providing a fermentation medium comprising acarbon substrate and triglycerides, diglycerides, monoglycerides, andphospholipids, or a mixture thereof; and contacting the fermentationmedium with an alcohol-producing microorganism comprising a butanolbiosynthetic pathway wherein said microorganism further comprises anengineered polynucleotide encoding a polypeptide having lipase activityand which expresses and secretes or displays the polypeptide such thatthe lipase activity is present in the fermentation medium. Inembodiments, the fermentation medium further comprises one or morecarboxylic acids. In embodiments, the carbon substrate is derived frombiomass. In embodiments, the biomass is corn or sugar cane. Inembodiments, the carbon substrate and the triglycerides diglycerides,monoglycerides, and phospholipids are derived from the same biomass.

Provided herein are fermentation media comprising an alcohol-producingmicroorganism comprising a butanol biosynthetic pathway and furthercomprising an engineered polynucleotide encoding a polypeptide havinglipase activity which is expressed and secreted or displayed, butylesters, and butanol.

Also provided are animal feed products comprising a microorganismsdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

The accompanying drawings and sequence listing, which are incorporatedherein and form a part of the specification, illustrate the presentinvention and, together with the description, further serve to explainthe principles of the invention and to enable a person skilled in thepertinent art to make and use the invention.

FIG. 1 schematically illustrates an exemplary method and system of thepresent invention, in which a microorganism is supplied to afermentation vessel along with carboxylic acid and/or native oil.

FIG. 2 depicts example biosynthetic pathways for biosynthesis ofisobutanol from pyruvate.

FIG. 3 is a map of plasmid pRS423::TEF1(M4)-CdLIP1 (“pNAK10”; SEQ ID NO:45; see Example 1), bearing the Candida deformans LIP1 lipase undertranscriptional control of the constitutive TEF1(M4) promoter (NevoigtE, Kohnke J, Fischer C R, Alper H, Stahl U, & Stephanopoulos G (2006),Engineering of promoter replacement cassettes for fine-tuning of geneexpression in Saccharomyces cerevisiae. Appl Environ Microbiol72:5266-5273) and the CYC1 transcriptional terminator, in a yeast-E.coli shuttle vector.

FIG. 4 is a map of plasmid pRS423::TEF1(M4)-THlip (“pTVAN2”; SEQ ID NO:100; see Example 2), bearing the Thermomyces lanuginosus Tlan lipaseunder transcriptional control of the constitutive TEF1(M4) promoter(Nevoigt E, et al.) and the CYC1 transcriptional terminator, in ayeast-E. coli shuttle vector.

FIG. 5 is a map of plasmid pRS423::TEF1(M4)-CalB (“pTVAN3”; SEQ ID NO:101; See Example 7), bearing the Candida antarctica CalB lipase undertranscriptional control of the constitutive TEF1(M4) promoter (NevoigtE, et al.) and the CYC1 transcriptional terminator, in a yeast-E. colishuttle vector.

FIG. 6 is a map of plasmid pYZ090ΔalsS (SEQ ID NO: 43; see Examples),which bears the ketol-acid reductoisomerase (KAR1) enzyme ORF in ayeast-E. coli shuttle vector.

FIG. 7. Map of plasmid pBP915 (SEQ ID NO: 44; see Examples 9 and 10),which bears the ORFs encoding the dihydroxyacid dehydratase enzyme andthe alcohol dehydrogenase enzyme in a yeast-E. coli shuttle vector.

SEQ ID NOs: 1 and 2 are nucleic acid and amino acid sequences for lipaseB (“CalB”) from Candida antarctica.

SEQ ID NOs: 3 and 4 are nucleic acid and amino acid sequences for lipase1 (“LIP1”) from Candida deformans.

SEQ ID NOs: 5 and 6 are nucleic acid and amino acid sequences for Tlanlipase (“Tlan”) from Thermomyces lanuginosus.

SEQ ID NOs: 255 and 256 are nucleic acid and amino acid sequences forlipase 3 (“lip3”) from Aspergillus tubingensis.

SEQ ID NOs: 7, 8, 9, and 257 are coding sequences for CalB, LIP1, Tlan,and lip3 lipases from Candida antarctica, Candida deformans, Thermomyceslanuginosus, and Aspergillus tubingensis, codon-optimized for expressionin S. cerevisiae.

SEQ ID NOs: 46 and 47 are nucleic acid and amino acid sequences for aCalB variant with the modification N99A.

SEQ ID NOs: 48 and 49 are nucleic acid and amino acid sequences for aLIP1 variant with the modification N146A.

SEQ ID NOs: 50 and 51 are nucleic acid and amino acid sequences for aLIP1 variant with the modification N167A.

SEQ ID NOs: 52 and 53 are nucleic acid and amino acid sequences for aLIP1 variant with the modifications N146A and N167A.

SEQ ID NOs: 54 and 55 are nucleic acid and amino acid sequences for aTlan variant with the modification N55A.

SEQ ID NOs: 271 and 272 are nucleic acid and amino acid sequences for alip3 variant with the modification N59A.

SEQ ID NOs: 273 and 274 are nucleic acid and amino acid sequences for alip3 variant with the modification N269A.

SEQ ID NOs: 275 and 276 are nucleic acid and amino acid sequences for alip3 variant with the modifications N59A and N269A.

SEQ ID NOs: 241 and 248 are amino acid sequences for lipases fromAspergillus kawachii, Aspergillus niger, Yarrowia lipolytica,Talaromyces thermophilus.

SEQ ID NOs: 249 and 254 are amino acid sequences of cell surface anchordomains of S. cerevisiae.

SEQ ID NOs: 258 and 259 are the amino acid sequences of alcoholdehydrogenase enzymes from Achromobacter xylosoxidans and Beijerinkiaindica.

SEQ ID NOs: 260 and 261 are the amino acid sequences of keto-aciddecarboxylases from Lactococcus lactis and Listeria grayi.

SEQ ID NOs: 262 and 263 are the amino acid sequences of dihydroxyaciddehdratases from Streptococcus mutans and Lactococcus lactis.

SEQ ID NOs: 10-45, 56-144, 153-238, 240, 264-270, and 278 are sequencesof synthetic constructs and primers described in the Examples.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application including the definitions will control. Also, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. All publications, patentsand other references mentioned herein are incorporated by reference intheir entireties for all purposes.

In order to further define this invention, the following terms anddefinitions are herein provided.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers. For example, a composition, a mixture, a process,a method, an article, or an apparatus that comprises a list of elementsis not necessarily limited to only those elements but can include otherelements not expressly listed or inherent to such composition, mixture,process, method, article, or apparatus. Further, unless expressly statedto the contrary, “or” refers to an inclusive or and not to an exclusiveor. For example, a condition A or B is satisfied by any one of thefollowing: A is true (or present) and B is false (or not present), A isfalse (or not present) and B is true (or present), and both A and B aretrue (or present).

Also, the indefinite articles “a” and “an” preceding an element orcomponent of the invention are intended to be nonrestrictive regardingthe number of instances, i.e., occurrences of the element or component.Therefore “a” or “an” should be read to include one or at least one, andthe singular word form of the element or component also includes theplural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdescribed in the application.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates orsolutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or to carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities. Inone embodiment, the term “about” means within 10% of the reportednumerical value, alternatively within 5% of the reported numericalvalue.

“Biomass” as used herein refers to a natural product containinghydrolysable polysaccharides that provide fermentable sugars, includingany sugars and starch derived from natural resources such as corn, sugarcane, wheat, cellulosic or lignocellulosic material and materialscomprising cellulose, hemicellulose, lignin, starch, oligosaccharides,disaccharides and/or monosaccharides, and mixtures thereof. Biomass mayalso comprise additional components, such as protein and/or lipids.Biomass may be derived from a single source, or biomass can comprise amixture derived from more than one source; for example, biomass maycomprise a mixture of corn cobs and corn stover, or a mixture of grassand leaves. Biomass includes, but is not limited to, bioenergy crops,agricultural residues, municipal solid waste, industrial solid waste,sludge from paper manufacture, yard waste, wood and forestry waste.Examples of biomass include, but are not limited to, corn grain, corncobs, crop residues such as corn husks, corn stover, grasses, wheat,rye, wheat straw, barley, barley straw, hay, rice straw, switchgrass,waste paper, sugar cane bagasse, sorghum, soy, components obtained frommilling of grains, trees, branches, roots, leaves, wood chips, sawdust,shrubs and bushes, vegetables, fruits, flowers, animal manure, andmixtures thereof. For example, mash or juice or molasses or hydrolysatemay be formed from biomass by any processing known in the art forprocessing the biomass for purposes of fermentation, such as by milling,treating and/or liquefying and comprises fermentable sugar and maycomprise an amount of water. For example, cellulosic and/orlignocellulosic biomass may be processed to obtain a hydrolysatecontaining fermentable sugars by any method known to one skilled in theart. A low ammonia pretreatment is disclosed in US Patent ApplicationPublication US20070031918A1, which is herein incorporated by reference.Enzymatic saccharification of cellulosic and/or lignocellulosic biomasstypically makes use of an enzyme consortium for breaking down celluloseand hemicellulose to produce a hydrolysate containing sugars includingglucose, xylose, and arabinose. (Saccharification enzymes suitable forcellulosic and/or lignocellulosic biomass are reviewed in Lynd, L. R.,et al. (Microbiol. Mol. Biol. Rev., 66:506-577, 2002).

Mash or juice or molasses or hydrolysate may include feedstock 12 andfeedstock slurry 16 as described herein. An aqueous feedstream may bederived or formed from biomass by any processing known in the art forprocessing the biomass for purposes of fermentation, such as by milling,treating and/or liquefying and comprises fermentable carbon substrate(eg. sugar) and water. An aqueous feedstream may include feedstock 12and feedstock slurry 16 as described herein.

“Product alcohol” as used herein refers to any alcohol that can beproduced by a microorganism in a fermentation process that utilizesbiomass as a source of fermentable carbon substrate. Product alcoholsinclude, but are not limited to, C₁ to C₅ alkyl alcohols. Inembodiments, the product alcohols are C₂ to C₅ alkyl alcohols. Inadditional embodiments, the product alcohols are C₂ to C₅ alkylalcohols. It will be appreciated that C₁ to C₅ alkyl alcohols include,but are not limited to, methanol, ethanol, propanol, butanol, andpentanol. Likewise C₂ to C₅ alkyl alcohols include, but are not limitedto, ethanol, propanol, butanol, and pentanol. “Alcohol” is also usedherein with reference to a product alcohol.

“Butanol” as used herein refers with specificity to the butanol isomers1-butanol (1-BuOH), 2-butanol (2-BuOH) and/or isobutanol (iBuOH ori-BuOH or I-BUOH, also known as 2-methyl-1-propanol), eitherindividually or as mixtures thereof.

“Propanol” as used herein refers to the propanol isomers isopropanol or1-propanol.

“Pentanol” as used herein refers to the pentanol isomers 1-pentanol,3-methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethyl-1-propanol,3-pentanol, 2-pentanol, 3-methyl-2-butanol, or 2-methyl-2-butanol.

“In Situ Product Removal (ISPR)” as used herein means the selectiveremoval of a specific fermentation product from a biological processsuch as fermentation to control the product concentration in thebiological process as the product is produced.

“Fermentable carbon source” or “fermentable carbon substrate” as usedherein means a carbon source capable of being metabolized by themicroorganisms disclosed herein for the production of fermentativealcohol. Suitable fermentable carbon sources include, but are notlimited to, monosaccharides, such as glucose or fructose; disaccharides,such as lactose or sucrose; oligosaccharides; polysaccharides, such asstarch or cellulose; one carbon substrates including methane; andmixtures thereof.

“Feedstock” as used herein means a feed in a fermentation process, thefeed containing a fermentable carbon source with or without undissolvedsolids, and where applicable, the feed containing the fermentable carbonsource before or after the fermentable carbon source has been liberatedfrom starch or obtained from the breakdown of complex sugars by furtherprocessing, such as by liquefaction, saccharification, or other process.Feedstock includes or is derived from a biomass. Suitable feedstocksinclude, but are not limited to, rye, wheat, corn, cane and mixturesthereof.

“Undissolved solids” as used herein means non-fermentable portions offeedstock, for example germ, fiber, and gluten.

“Fermentation broth” as used herein means the mixture of water, sugars,dissolved solids, microorganisms producing alcohol, product alcohol andall other constituents of the material held in the fermentation vesselin which product alcohol is being made by the reaction of sugars toalcohol, water and carbon dioxide (CO₂) by the microorganisms present.

From time to time, as used herein the term “fermentation medium” and“fermented mixture” can be used synonymously with “fermentation broth”.

“Fermentation vessel” as used herein means the vessel in which thefermentation reaction by which product alcohol such as butanol is madefrom sugars is carried out.

The term “effective titer” as used herein, refers to the total amount ofa particular alcohol (e.g., butanol) produced by fermentation or alcoholequivalent of the alcohol ester produced by alcohol esterification perliter of fermentation medium. For example, the effective titer ofbutanol in a unit volume of a fermentation includes: (i) the amount ofbutanol in the fermentation medium; (ii) the amount of butanol recoveredfrom the organic extractant; (iii) the amount of butanol recovered fromthe gas phase, if gas stripping is used, and (iv) the alcohol equivalentof the butanol ester in either the organic or aqueous phase.

“Saccharification” as used herein means the break down ofoligosaccharides into monosaccharides. “Simultaneous saccharificationand fermentation” means fermentation and saccharification occurconcurrently in the same vessel.

As used herein, “saccharification enzyme” means one or more enzymes thatare capable of hydrolyzing polysaccharides and/or ologosaccharides, e.g,alpha-1,4-glucosidic bonds of glycogen, starch. Saccharification enzymesmay include enzymes capable of hydrolyzing cellulosic or lignocellulosicmaterials as well.

As used herein, “lipase activity” means the enzymatic activity ofcatalyzing the hydrolysis of ester chemical bonds in water-insoluble orpoorly water soluble lipid substrates. Lipases are a subclass of theesterases, and as such, “lipase activity” also means the enzymaticactivity of catalyzing the hydrolysis of an ester into a carboxylic acidand an alcohol, and, as used herein, “lipase activity” also means theenzymatic activity of esterifying alcohol and carboxylic acid into analcohol ester of a carboxylic acid.

As used herein, “glycosylation” is the enzymatic addition ofcarbohydrate molecules to biological macromolecules such as proteins,which can occur when proteins are targeted for secretion out of thecell. In O-glycosylation of proteins, the carbohydrates are attached tothe hydroxyl groups of serine, threonine, or tyrosine residues. InN-glycosylation of proteins, the carbohydrates are attached to the amideside chain of asparagine (N) residues in the consensus sequence NXS/T,where X is any amino acid and S/T is serine or threonine. “Glycosylated”as used herein refers to a protein molecule with carbohydratescovalently attached.

“Liquefaction vessel” as used herein means the vessel in whichliquefaction is carried out. Liquefaction is the process in whicholigosaccharides are liberated from the feedstock. In embodiments wherethe feedstock is corn, oligosaccharides are liberated from the cornstarch content during liquefaction.

The term “separation” as used herein is synonymous with “recovery” andrefers to removing a chemical compound from an initial mixture to obtainthe compound in greater purity or at a higher concentration than thepurity or concentration of the compound in the initial mixture.

The terms “water-immiscible” or “insoluble” refer to a chemicalcomponent, such as an extractant or solvent, which is incapable ofmixing with an aqueous solution, such as a fermentation broth, in such amanner as to form one liquid phase.

“Extractant” or “ISPR extractant” as used herein means an organicsolvent used to extract any product alcohol such as butanol, or used toextract any product alcohol ester produced by a catalyst from a productalcohol and a carboxylic acid or lipid. From time to time, as usedherein the term “solvent” may be used synonymously with “extractant”.For the processes described herein, extractants are water-immiscible.

“Native oil” as used herein refers to lipids obtained from plants (e.g.,biomass) or animals. “Plant-derived oil” as used herein refers to lipidsobtained from plants in particular. From time to time, “lipids” may beused synonymously with “oil” and “acyl glycerides.” Native oils include,but are not limited to, tallow, corn, canola, capric/caprylictriglycerides, castor, coconut, cottonseed, fish, jojoba, lard, linseed,neetsfoot, oiticica, palm, peanut, rapeseed, rice, safflower, soya,sunflower, tung, jatropha and vegetable oil blends.

The term “organic phase”, as used herein, refers to the non-aqueousphase of a biphasic mixture obtained by contacting a fermentation brothwith a water-immiscible organic extractant.

The term “fatty acid” as used herein refers to a carboxylic acid (e.g.,aliphatic monocarboxylic acid) having C₄ to C₂₈ carbon atoms (mostcommonly C₁₂ to C₂₄ carbon atoms), which is either saturated orunsaturated. Fatty acids may also be branched or unbranched. Fatty acidsmay be derived from, or contained in esterified form, in an animal orvegetable fat, oil, or wax. Fatty acids may occur naturally in the formof glycerides in fats and fatty oils or may be obtained by hydrolysis offats or by synthesis. The term fatty acid may describe a single chemicalspecies or a mixture of fatty acids. Fatty acids may comprise a mixtureof both protonated and unprotonated fatty acids, wherein theunprotonated fatty acids are salts (e.g., sodium, potassium, ammonium orcalcium ion salts) of unprotonated fatty acids. In addition, the termfatty acid also encompasses free fatty acids.

The term “fatty alcohol” as used herein refers to an alcohol having analiphatic chain of C₄ to C₂₂ carbon atoms, which is either saturated orunsaturated.

The term “fatty aldehyde” as used herein refers to an aldehyde having analiphatic chain of C₄ to C₂₂ carbon atoms, which is either saturated orunsaturated.

The term “carboxylic acid” as used herein refers to any organic compoundwith the general chemical formula —COOH in which a carbon atom is bondedto an oxygen atom by a double bond to make a carbonyl group (—C═O) andto a hydroxyl group (—OH) by a single bond. A carboxylic acid may be inthe form of the protonated carboxylic acid, or in the form of a salt ofa carboxylic acid (for example, an ammonium, sodium or potassium salt),or as a mixture of protonated carboxylic acid and salt of a carboxylicacid. The term carboxylic acid may describe a single chemical species(e.g., oleic acid), or a mixture of carboxylic acids as can be produced,for example, by the hydrolysis of biomass-derived fatty-acid esters ortriglycerides, diglycerides, monoglyerides and phopholipids.

The term “butanol biosynthetic pathway” or “butanol production pathway”as used herein refers to an enzyme pathway to produce 1-butanol,2-butanol, or isobutanol.

The term “1-butanol biosynthetic pathway” or “1-butanol productionpathway” as used herein refers to an enzyme pathway to produce 1-butanolfrom acetyl-coenzyme A (acetyl-CoA).

The term “2-butanol biosynthetic pathway” or “2-butanol productionpathway” as used herein refers to an enzyme pathway to produce 2-butanolfrom pyruvate.

The term “isobutanol biosynthetic pathway” or “isobutanol productionpathway” as used herein refers to an enzyme pathway to produceisobutanol from pyruvate.

The term “alcohol biosynthetic pathway” or “alcohol production pathway”as used herein refers to an enzymatic pathway to convert a carbonsubstrate to an alcohol. A recombinant host cell comprising an“engineered alcohol production pathway” refers to a host cell containinga modified pathway that produces alcohol in a manner different than thatnormally present in the host cell. Such differences include productionof an alcohol not typically produced by the host cell, or increased ormore efficient production.

The term “gene” refers to a nucleic acid fragment that is capable ofbeing expressed as a specific protein, optionally including regulatorysequences preceding (5′ non-coding sequences) and following (3′non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene (i.e, it is modifiedfrom its native state or is from another source), comprising regulatoryand coding sequences that are not found together in nature. Accordingly,a chimeric gene may comprise regulatory sequences and coding sequencesthat are derived from different sources, or regulatory sequences andcoding sequences derived from the same source, but arranged in a mannerdifferent than that found in nature. “Endogenous gene” refers to anative gene in its natural location in the genome of an organism. A“foreign gene” or “heterologous gene” refers to a gene not normallyfound as a native gene in the host organism, but that is introduced intothe host organism by gene transfer. Foreign genes can comprise nativegenes inserted into a non-native organism, or chimeric genes.

As used herein the term “coding region” refers to a DNA sequence thatcodes for a specific amino acid sequence. “Suitable regulatorysequences” refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing sites, effectorbinding sites and stem-loop structures.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to a nucleic acidmolecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA).As used herein, a “gene” is a polynucleotide. A polynucleotide cancontain the nucleotide sequence of the full-length gene or cDNAsequence, or a fragment thereof, including the untranslated 5′ and 3′sequences and the coding sequences. The polynucleotide can be composedof any polyribonucleotide or polydeoxyribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA (e.g. heterologous DNA).For example, polynucleotides can be composed of single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions.“Polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

“Engineered polynucleotide” as used herein refers to a polynucleotidethat has been modified from a form found in nature or that is introducedinto a host organism by gene transfer such as by transformation. Suchmodification includes, for example, linking two sequences not foundlinked in nature, such as operably linking a coding sequence with apromoter not found operably linked with the coding sequence in nature,or linking two coding sequences together to create a chimeric codingsequence. Such modification also includes creating one or morenucleotide changes, including base substitutions, insertions, ordeletions, to a polynucleotide found in nature.

A polynucleotide sequence may be referred to as “isolated,” in which ithas been removed from its native environment. For example, aheterologous polynucleotide encoding a polypeptide or polypeptidefragment having dihydroxy-acid dehydratase activity contained in avector is considered isolated for the purposes of the present invention.Further examples of an isolated polynucleotide include recombinantpolynucleotides maintained in heterologous host cells or purified(partially or substantially) polynucleotides in solution. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. An isolatedpolynucleotide fragment in the form of a polymer of DNA may be comprisedof one or more segments of cDNA, genomic DNA or synthetic DNA.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

As used herein, “recombinant microorganism” refers to microorganisms,such as bacteria or yeast, that are modified by use of recombinant DNAtechniques, such as by engineering a host cell to comprise abiosynthetic pathway such as butanol.

As used herein the term “codon degeneracy” refers to the nature in thegenetic code permitting variation of the nucleotide sequence withouteffecting the amino acid sequence of an encoded polypeptide. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA. Such optimizationincludes replacing at least one, or more than one, or a significantnumber, of codons with one or more codons that are more frequently usedin the genes of that organism.

Deviations in the nucleotide sequence that comprise the codons encodingthe amino acids of any polypeptide chain allow for variations in thesequence coding for the gene. Since each codon consists of threenucleotides, and the nucleotides comprising DNA are restricted to fourspecific bases, there are 64 possible combinations of nucleotides, 61 ofwhich encode amino acids (the remaining three codons encode signalsending translation). The “genetic code” which shows which codons encodewhich amino acids is reproduced herein as Table 1. As a result, manyamino acids are designated by more than one codon. For example, theamino acids alanine and proline are coded for by four triplets, serineand arginine by six, whereas tryptophan and methionine are coded by justone triplet. This degeneracy allows for DNA base composition to varyover a wide range without altering the amino acid sequence of theproteins encoded by the DNA.

TABLE 1 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S) TATTyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y) TGC TTA Leu (L)TCA Ser (S) TAA Stop TGA Stop TTG Leu (L) TCG Ser (S) TAG Stop TGG Trp(W) C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC Leu (L) CCCPro (P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P) CAA Gln (Q) CGAArg (R) CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R) A ATT Ile (I)ACT Thr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I) ACC Thr (T) AAC Asn (N)AGC Ser (S) ATA Ile (I) ACA Thr (T) AAA Lys (K) AGA Arg (R) ATG Met (M)ACG Thr (T) AAG Lys (K) AGG Arg (R) G GTT Val (V) GCT Ala (A) GAT Asp(D) GGT Gly (G) GTC Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val(V) GCA Ala (A) GAA Glu (E) GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu(E) GGG Gly (G)

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing peptide chain. Codonpreference, or codon bias, differences in codon usage between organisms,is afforded by degeneracy of the genetic code, and is well documentedamong many organisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, it is possible to calculate therelative frequencies of codon usage. Codon usage tables are readilyavailable, for example, at the “Codon Usage Database” available athttp://www.kazusa.or.jp/codon/ (visited Mar. 20, 2008), and these tablescan be adapted in a number of ways. See Nakamura, Y., et al. Nucl. AcidsRes. 28:292 (2000). Codon usage tables for yeast, calculated fromGenBank Release 128.0 [15 Feb. 2002], are reproduced below as Table 2.This table uses mRNA nomenclature, and so instead of thymine (T) whichis found in DNA, the tables use uracil (U) which is found in RNA. Table2 has been adapted so that frequencies are calculated for each aminoacid, rather than for all 64 codons.

TABLE 2 Codon Usage Table for Saccharomyces cerevisiae Genes Frequencyper Amino Acid Codon Number thousand Phe UUU 170666 26.1 Phe UUC 12051018.4 Leu UUA 170884 26.2 Leu UUG 177573 27.2 Leu CUU 80076 12.3 Leu CUC35545 5.4 Leu CUA 87619 13.4 Leu CUG 68494 10.5 Ile AUU 196893 30.1 IleAUC 112176 17.2 Ile AUA 116254 17.8 Met AUG 136805 20.9 Val GUU 14424322.1 Val GUC 76947 11.8 Val GUA 76927 11.8 Val GUG 70337 10.8 Ser UCU153557 23.5 Ser UCC 92923 14.2 Ser UCA 122028 18.7 Ser UCG 55951 8.6 SerAGU 92466 14.2 Ser AGC 63726 9.8 Pro CCU 88263 13.5 Pro CCC 44309 6.8Pro CCA 119641 18.3 Pro CCG 34597 5.3 Thr ACU 132522 20.3 Thr ACC 8320712.7 Thr ACA 116084 17.8 Thr ACG 52045 8.0 Ala GCU 138358 21.2 Ala GCC82357 12.6 Ala GCA 105910 16.2 Ala GCG 40358 6.2 Tyr UAU 122728 18.8 TyrUAC 96596 14.8 His CAU 89007 13.6 His CAC 50785 7.8 Gln CAA 178251 27.3Gln CAG 79121 12.1 Asn AAU 233124 35.7 Asn AAC 162199 24.8 Lys AAA273618 41.9 Lys AAG 201361 30.8 Asp GAU 245641 37.6 Asp GAC 132048 20.2Glu GAA 297944 45.6 Glu GAG 125717 19.2 Cys UGU 52903 8.1 Cys UGC 310954.8 Trp UGG 67789 10.4 Arg CGU 41791 6.4 Arg CGC 16993 2.6 Arg CGA 195623.0 Arg CGG 11351 1.7 Arg AGA 139081 21.3 Arg AGG 60289 9.2 Gly GGU156109 23.9 Gly GGC 63903 9.8 Gly GGA 71216 10.9 Gly GGG 39359 6.0 StopUAA 6913 1.1 Stop UAG 3312 0.5 Stop UGA 4447 0.7

By utilizing this or similar tables, one of ordinary skill in the artcan apply the frequencies to any given polypeptide sequence, and producea nucleic acid fragment of a codon-optimized coding region which encodesthe polypeptide, but which uses codons optimal for a given species.

Randomly assigning codons at an optimized frequency to encode a givenpolypeptide sequence, can be done manually by calculating codonfrequencies for each amino acid, and then assigning the codons to thepolypeptide sequence randomly. Additionally, various algorithms andcomputer software programs are readily available to those of ordinaryskill in the art. For example, the “EditSeq” function in the LasergenePackage, available from DNAstar, Inc., Madison, Wis., thebacktranslation function in the VectorNTI Suite, available fromInforMax, Inc., Bethesda, Md., and the “backtranslate” function in theGCG-Wisconsin Package, available from Accelrys, Inc., San Diego, Calif.In addition, various resources are publicly available to codon-optimizecoding region sequences, e.g., the “backtranslation” function athttp://www.entelechon.com/bioinformatics/backtranslation.php?lang=eng(visited Apr. 15, 2008) and the “backtranseq” function available athttp://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html (visited Jul. 9, 2002).Constructing a rudimentary algorithm to assign codons based on a givenfrequency can also easily be accomplished with basic mathematicalfunctions by one of ordinary skill in the art.

Codon-optimized coding regions can be designed by various methods knownto those skilled in the art including software packages such as“synthetic gene designer” (userpages.umbc.edu/˜wug1/codon/sgd/, accessedMar. 19, 2012_).

A polynucleotide or nucleic acid fragment is “hybridizable” to anothernucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule,when a single-stranded form of the nucleic acid fragment can anneal tothe other nucleic acid fragment under the appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known and exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989),particularly Chapter 11 and Table 11.1 therein. The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. Stringency conditions can be adjusted to screen formoderately similar fragments (such as homologous sequences fromdistantly related organisms), to highly similar fragments (such as genesthat duplicate functional enzymes from closely related organisms).Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6×SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65°C. An additional set of stringent conditions include hybridization at0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1% SDS followed by0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of T_(m) for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherT_(m)) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., supra, 9.50-9.51). For hybridizations with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7-11.8). In one embodimentthe length for a hybridizable nucleic acid is at least about 10nucleotides. Preferably a minimum length for a hybridizable nucleic acidis at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least about 30nucleotides. Furthermore, the skilled artisan will recognize that thetemperature and wash solution salt concentration may be adjusted asnecessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as Basic Local Alignment SearchTool (“BLAST”; Altschul, S. F., et al., J. Mol. Biol., 215:403-410(1993)). In general, a sequence of ten or more contiguous amino acids orthirty or more nucleotides is necessary in order to putatively identifya polypeptide or nucleic acid sequence as homologous to a known proteinor gene. Moreover, with respect to nucleotide sequences, gene specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to specifically identify and/or isolatea nucleic acid fragment comprising the sequence. The instantspecification teaches the complete amino acid and nucleotide sequenceencoding particular proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Accordingly, the instant invention comprises the completesequences as provided herein, as well as substantial portions of thosesequences as defined above.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those disclosed in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the MegAlign™ program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesis performed using the “Clustal method of alignment” which encompassesseveral varieties of the algorithm including the “Clustal V method ofalignment” corresponding to the alignment method labeled Clustal V(disclosed by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in theMegAlign™ program of the LASERGENE bioinformatics computing suite(DNASTAR Inc.). For multiple alignments, the default values correspondto GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters forpairwise alignments and calculation of percent identity of proteinsequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2,GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of thesequences using the Clustal V program, it is possible to obtain a“percent identity” by viewing the “sequence distances” table in the sameprogram. Additionally the “Clustal W method of alignment” is availableand corresponds to the alignment method labeled Clustal W (described byHiggins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al.,Comput. Appl. Biosci. 8:189-191 (1992)) and found in the MegAlign™ v6.1program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.).Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTHPENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5,Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). Afteralignment of the sequences using the Clustal W program, it is possibleto obtain a “percent identity” by viewing the “sequence distances” tablein the same program.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, includingvariants or polypeptides from other species, wherein such polypeptideshave the same or similar function or activity. Useful examples ofpercent identities include, but are not limited to: 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100%may be useful in describing the present invention, such as 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99%. Suitable nucleic acid fragments not only have the above homologiesbut typically encode a polypeptide having at least 50 amino acids,preferably at least 100 amino acids, more preferably at least 150 aminoacids, still more preferably at least 200 amino acids, and mostpreferably at least 250 amino acids.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.,215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L.and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987). Additional methods usedhere are in Methods in Enzymology, Volume 194, Guide to Yeast Geneticsand Molecular and Cell Biology (Part A, 2004, Christine Guthrie andGerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.).

The genetic manipulations of a recombinant host cell disclosed hereincan be performed using standard genetic techniques and screening and canbe made in any host cell that is suitable to genetic manipulation(Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp. 201-202). In embodiments, the recombinanthost cell is E. coli. In embodiments, a recombinant host cell disclosedherein can be any yeast or fungal host useful for genetic modificationand recombinant gene expression. In other embodiments, a recombinanthost cell can be a member of the genera Zygosaccharomyces,Schizosaccharomyces, Dekkera, Issatchenkia, Torulopsis, Brettanomyces,Torulaspora, Hanseniaspora, Kluyveromyces, and some species of Candida.In another embodiment, a recombinant host cell can be Saccharomycescerevisiae.

The Applicants have discovered that recombinant host cells which areable to express and secrete lipase enzymes into a fermentation mediumproduce a catalyst that will catalyze the esterification of alcohol andcarboxylic acid. Such host cells represent an improvement to host cellsused in fermentative production of alcohols because the esterificationof the alcohol may allow the cells to produce alcohol with greaterefficiency, or to produce an amount of alcohol in excess of the amountof alcohol that would exert a toxic effect on the host cells. Also, useof such recombinant microorganisms can reduce or eliminate the need toadd purified lipase enzyme to a fermentation medium to carry out theprocesses described herein, which may provide cost and operationaladvantages.

Furthermore, fermentative production of alcohols typically utilizes arenewable biomass feedstock to supply the carbon substrate which arecombinant microorganism converts to product alcohol. Such feedstockscan contain an amount of triglycerides. When extractive fermentation ispracticed to remove the product alcohol from the fermentation, thetriglycerides may build up over time, decreasing the partitioncoefficient and recyclability of the extractant. The lipases secreted bythe recombinant host cells provided herein can advantageously hydrolyzethe triglycerides into free fatty acids which may be substrates foresterification and which may also have less effect on the partitioncoefficient of an extractant for product alcohols.

Polypeptides Having Lipase Activity

Recombinant host cells disclosed herein comprise polynucleotides havingpolypeptides having lipase activity. Examples of lipase polynucleotidesand polypeptides and the organisms from which they are derived areprovided in Table 3.

TABLE 3 Example lipase polynucleotides and polypeptides Nucleic AcidSequence, Codon- Species and optimized for Accession Number Nucleic acidAmino acid expression in or Reference SEQ ID NO: SEQ ID NO: S.cerevisiae Candida antarctica 1 2 7 Z30645 Candida deformans 3 4 8AJ428393 Thermomyces 5 6 9 lanuginosus AF054513 Aspergillus 255 256 257tubingensis lip3 U.S. Pat. No. 7,371,423B2; PCT App. Pub. No. WO98/45453

BLAST analysis of the non-redundant protein sequence database at theNational Center for Biotechnology Information was performed using, asquery sequences, lipases described in Table 3, in order to identifyproteins with high (>90%) sequence similarity. Results are shown inTable 4 (information retrieved from the non-redundant protein sequencedatabase online at the National Center for Biotechnology Information onJan. 22, 2012). While proteins with sequence similarity greater than 90%are considered to be lipases that are predicted to perform similarly tolipases described herein, sequences with similarity as low as ˜30% tothe query sequences are annotated as lipases and are contemplated foruse with the methods and compositions described herein.

TABLE 4 Additional example lipase polypeptides Amino GenBank acidAccession SEQ Source Organism Number Identity to lipase ID NO:Aspergillus GAA84811 99% to Aspergillus 241 kawachii tubingensisAspergillus niger BAL22280 98% to Aspergillus 242 tubingensisAspergillus niger XP_001397501 93% to Aspergillus 243 tubingensisAspergillus niger ABG73613 93% to Aspergillus 244 tubingensisAspergillus niger ABG37906 93% to Aspergillus 245 tubingensis Yarrowialipolytica XP_500282 92% to Candida 246 deformans Yarrowia lipolyticaADL57415 91% to Candida 247 deformans Talaromyces AEE61324 90% toThermomyces 248 thermophiles lanuginosus

In addition to the lipases described above and in Tables 3 and 4,suitable lipase sequences may be derived from any source, including, forexample, Absidia, Achromobacter, Aeromonas, Alcaligenes, Alternaria,Aspergillus, Achromobacter, Aureobasidium, Bacillus, Beauveria,Brochothrix, Candida, Chromobacter, Coprinus, Fusarium, Geotricum,Hansenula, Humicola, Hyphozyma, Lactobacillus, Metarhizium, Mucor,Nectria, Neurospora, Paecilomyces, Penicillium, Pseudomonas,Rhizoctonia, Rhizomucor, Rhizopus, Rhodosporidium, Rhodotorula,Saccharomyces, Sus, Sporobolomyces, Thermomyces, Thiarosporella,Trichoderma, Verticillium, and/or a strain of Yarrowia. In embodiments,the source of the lipase is selected from the group consisting ofAbsidia blakesleena, Absidia corymbifera, Achromobacter iophagus,Alcaligenes sp., Alternaria brassiciola, Aspergillus flavus, Aspergillusniger, Aspergillus kawachii, Aspergillus tubingensis, Aureobasidiumpullulans, Bacillus pumilus, Bacillus strearothermophilus, Bacillussubtilis, Brochothrix thermosohata, Candida cylindracea (Candidarugosa), Candida paralipolytica, Candida antarctica lipase A, Candidaantarctica lipase B, Candida ernobii, Candida deformans, Candidathermophila, Chromobacter viscosum, Coprinus cinerius, Fusariumoxysporum, Fusarium solani, Fusarium solani pisi, Fusarium roseumculmorum, Geotrichum candidum, Geotricum penicillatum, Hansenulaanomala, Humicola brevispora, Humicola brevis var. thermoidea, Humicolainsolens, Lactobacillus curvatus, Rhizopus niveus, Rhizopus oryzae,Penicillium cyclopium, Penicillium crustosum, Penicillium expansum,Penicillium sp. I, Penicillium sp. II, Pseudomonas aeruginosa,Pseudomonas alcaligenes, Pseudomonas cepacia (syn. Burkholderiacepacia), Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonasmaltophilia, Pseudomonas mendocina, Pseudomonas mephitica lipolytica,Pseudomonas alcaligenes, Pseudomonas plantari, Pseudomonaspseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri, andPseudomonas wisconsinensis, Rhizoctonia solani, Rhizomucor miehei,Rhizopus japonicus, Rhizopus microsporus, Rhizopus nodosus,Rhodosporidium toruloides, Rhodotorula glutinis, Saccharomycescerevisiae, Sporobolomyces shibatanus, Sus scrofa, Talaromycesthermophiles, Thermomyces lanuginosus (formerly Humicola lanuginose),Thiarosporella phaseolina, Trichoderma harzianum, Trichoderma reesei,and Yarrowia lipolytica. In embodiments, the lipase is selected from thegroup consisting of Thermomyces lanuginosus lipase, Aspergillus sp.lipase, Aspergillus niger lipase, Aspergillus tubingensis lip3, Candidaantarctica lipase B, Pseudomonas sp. lipase, Penicillium roquefortilipase, Penicillium camembertii lipase, Mucor javanicus lipase,Burkholderia cepacia lipase, Alcaligenes sp. lipase, Candida rugosalipase, Candida parapsilosis lipase, Candida deformans lipases, lipasesA and B from Geotrichum candidum, Neurospora crassa lipase, Nectriahaematococca lipase, Fusarium heterosporum lipase Rhizopus delemarlipase, Rhizomucor miehei lipase, Rhizopus arrhizus lipase, and Rhizopusoryzae lipase.

One of skill in the art will appreciate that polynucleotide sequencesthat encode polypeptides with lipase activity such as the polynucleotidesequences in the table above or derived from the indicated sources canbe codon-optimized for the recombinant host cell. Further, one of skillin the art will appreciate that truncations and conservativesubstitutions can be made to the polypeptide sequences given withouteliminating the lipase activity of the polypeptide. Accordingly,provided herein are polypeptides having at least about 75%, at leastabout 80%, at least about 90%, at least about 95%, at least about 97%,or at least about 99% identity to the sequences provided and activefragments thereof. Also provided are polynucleotides encoding suchpolypeptides.

For embodiments of the methods and host cells described herein, that thepolypeptide having lipase activity may be expressed and secreted by themicroorganism such that the lipase has activity in the fermentationmedium during the production of a product alcohol. One of skill in theart will appreciate that polypeptides expressed on the surface of amicroorganism, such as cell wall proteins which are processed throughthe secretory pathway, will be considered to be secreted since theactivity of a polypeptide expressed on the cell surface can be availableexternal to the cell. Thus, in embodiments, the secreted lipase isexpressed on the surface of the microorganism. Surface expression ofproteins is known in the art, as is modification of polypeptides totarget them for surface expression. (Washida, M., S. Takahashi, M. Uedaand A. Tanaka (2001). “Spacer-mediated display of active lipase on theyeast cell surface.” Appl Microbiol Biotechnol 56(5-6): 681-686,Matsumoto, T., H. Fukuda, M. Ueda, A. Tanaka and A. Kondo (2002).“Construction of yeast strains with high cell surface lipase activity byusing novel display systems based on the Flo1p flocculation functionaldomain.” Appl Environ Microbiol 68(9): 4517-4522, Mormeneo, M., I.Andres, C. Bofill, P. Diaz and J. Zueco (2008). “Efficient secretion ofBacillus subtilis lipase A in Saccharomyces cerevisiae by translationalfusion to the Pir4 cell wall protein.” Appl. Microbiol. Biotechnol.80(3): 437-445, Liu, W., H. Zhao, B. Jia, L. Xu and Y. Yan (2010).“Surface display of active lipase in Saccharomyces cerevisiae using Cwp2as an anchor protein.” Biotechnology Letters 32(2): 255-260, Su, G.-d.,X. Zhang and Y. Lin (2010). “Surface display of active lipase in Pichiapastoris using Sed1 as an anchor protein.” Biotechnology Letters 32(8):1131-1136, Kuroda, K. and M. Ueda (2011). “Cell surface engineering ofyeast for applications in white biotechnology.” Biotechnology Letters33(1): 1-9). In embodiments, a polypeptide provided herein is fused to adomain of a protein which targets the polypeptide to the cell surface Inembodiments, polypeptides provided herein are fused to a domain ofFlo1p, Pir4, Sed1, Sag1p, Cwp2, or Aga2. In embodiments, polypeptidesprovided herein are fused to a protein, or a fragment of a protein,having a GPI anchor motif. GPI anchor motifs are known to those of skillin the art and can be predicted by bioinformatics, for example by usingprediction engines (for example, the prediction engine online atmendel.imp.ac.at/gpi/fungi_server.html, accessed Mar. 19, 2012).(Eisenhaber B., et al. “A sensitive predictor for potential GPI lipidmodification sites in fungal protein sequences and its application togenome-wide studies for Aspergillus nidulans, Candida albicans,Neurospora crassa, Saccharomyces cerevisiae, and Schizosaccharomycespombe” J Mol. Biol. 2004 Mar. 19; 337(2):243-53.) Example polypeptidesequence domains which may be used target a polypeptide to the cellsurface of Saccharomyces cerevisiae are shown in Table 5. Systematicnames of the proteins in Table 5 are according to the SaccharomycesGenome Database (“SGD”; online at www.yeastgenome.org/; informationretrieved Mar. 13, 2012). One of skill in the art, equipped with thisdisclosure, will be able to use the example polypeptide sequences andother such sequences known in the art to construct polypeptides whichtarget lipase activity to the cell surface of a recombinantmicroorganism.

TABLE 5 Polypeptide sequences of cell surface anchor domains of S.cerevisiae proteins for surface display. Codons of nucleic Amino acidacid sequence sequence of SGD systematic corresponding to domain Proteinname name of protein protein domain SEQ ID NO: Sag1 YJR004C 331-650 249Aga2 YGL032C  1-87^(†) 250 Flo1 YAR050W  1-1099 251 Cwp2 YKL096W  1-92252 Sed1 YDR077W  2-338 253 ^(†)Co-express Aga2 domain with Aga1 (SGDsystematic name: YNR044W; SEQ ID NO: 254)

In embodiments, the lipase polypeptide sequences provided herein may bemodified such that glycosylation, including, but not limited to,N-glycosylation, reduced or eliminated. Such modification can be carriedout by mutating the polynucleotide encoding the polypeptide such thatone or more glycosylation motifs is removed. In embodiments, theglycosylation motif is an N-glycosylation motif. In embodiments, theglycosylation motif is NXS/T. In embodiments, the polypeptide havinglipase activity does not contain the glycosylation motif NXS/T.

Glycosylation can be reduced or eliminated by any means known in theart. For example, inhibitors of glycosylation such as tunicamycin may beemployed or the glycosylation mechanism in a host cell may be altered.Also, glycosylation motifs can be removed by site-directed mutagenesisusing techniques known in the art. For example, site-directedmutagenesis can be carried out using commercially available kits (forexample, the QuikChange II XL site directed mutagenesis kit, Catalog#200524, Stratagene, La Jolla, Calif.). Site-direct mutagenesis can becarried out by the method of Kunkel, involving incorporation of uracilinto the template to be mutated (Kunkel T A (1985) Rapid and efficientsite-specific mutagenesis without phenotypic selection. Proc. Natl.Acad. Sci. USA 82:488-492), or by the method of phosphorothioateincorporation (Taylor J W, Ott J, & Eckstein F (1985), The rapidgeneration of oligonucleotide-directed mutations at high frequency usingphosphorothioate-modified DNA. Nucleic Acids Res 13:8765-8785), or byother methods, in vitro and in vivo, known in the art. Primer design fortarget sites for mutagenesis is well-known in the art, and sequenceanalysis such as multiple sequence alignment to identify target sitesfor mutagenesis is likewise well-known.

In embodiments, mutagenesis is carried out such that the N of the motifis substituted with any other naturally occurring amino acid (A, R, D,C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V; see Table 1). Inembodiments, the N of the motif is substituted with A. In embodiments,mutagenesis is carried out such that the S/T of the motif is replacedwith any other naturally occurring amino acid (A, R, N, D, C, E, Q, G,H, I, L, K, M, F, P, W, Y, or V; see Table 1). In embodiments, both theN and the S/T are replaced with any other naturally occurring amino acid(A, R, J, D, C, E, Q, G, H, I, L, K, M, F, P, W, Y, or V, or S or T atthe N residue; A, R, J, D, C, E, Q, G, H, I, L, K, M, F, P, W, Y, or V,S or N at a T residue; A, R, J, D, C, E, Q, G, H, I, L, K, M, F, P, W,Y, or V, or T or N at an S residue). In embodiments, the glycosylationmotif NXS/T is replaced with the motif AXS/T.

In one non-limiting example, C. deformans contains two glycosylationsequences, NIS at codon 146 and NNT at 167. In embodiments, one or bothof those glycosylation sites is targeted for substitution and theindicated glycosylation sites are replaced with AIS and ANT,respectively. C. antarctica has NDT at 99, and T. lanuginosus has NIT at55. In embodiments, the indicated glycosylation sites are mutated suchthat the sequences are ADT and AIT at the indicated positions.

Given in Table 6 are predicted glycosylation sites lipase open readingframes from C. deformans, C. antarctica, and T. lanuginosus, andexamples of mutations that abolish those sites. The first column liststhe position in the polypeptide at which the glycosylation site occurs.The second column gives the glycosylation sequence at that position, andthe DNA sequence encoding it in the codon-optimized polynucleotide. Thethird column gives the polypeptide sequence at that position aftermutagenesis, and the DNA sequence required to effect that amino acidchange.

TABLE 6 Predicted glycosylation sites SEQ ID SEQ ID Yeast NO: of NO: ofspecies and nucleic amino glycosylation acid acid site se- se- positionNative Modified quence quence C. deformans  N I S A I S  48  49 146AATATCAGT GCTATCAGT C. deformans  N N T A N T  50  51 167 ACAATACATGCTATACAT C. antarctica  N D T A D T  46  47 99 AATGATACT GCTGATACTT. lanuginosus  N I T A I T  54  55 55 AACATTACA GCTATTACAA. tubingensis  N L T A L T 271 272 59 AACTTAACA GCTTTAACAA. tubingensis  N S T A S T 273 274 269 AATTCTACA GCTTCTACA

In addition, the nucleic acid and amino acid sequences of Candidadeformans lipase with both of the modifications listed in Table 6 (N146Aand N167A) are given as SEQ ID NOs: 52 and 53. The nucleic acid andamino acid sequences of an A. tubingensis lipase with both of themodifications listed in Table 6 (N59A and N269A) are given as SEQ IDNOs: 275 and 276.

As shown in the Examples, using techniques known in the art and/orprovided herein, one of skill in the art can readily modifyglycosylation motifs in lipases and determine the activity of suchlipases in methods and compositions provided herein.

One of skill in the art will appreciate that provided herein arepolypeptides having at least about 75%, at least about 80%, at leastabout 90%, at least about 95%, at least about 97%, or at least about 99%identity to the sequences provided and active fragments thereof. Alsoprovided are polynucleotides encoding such polypeptides. One of skill inthe art will also appreciate that active variants of the sequencesprovided herein can be created using techniques known in the art and ordescribed herein for use in the methods and compositions describedherein.

Recombinant Microorganisms and Butanol Biosynthetic Pathways

While not wishing to be bound by theory, it is believed that theimprovements and processes described herein may be useful in conjunctionwith any alcohol producing microorganism, particularly recombinantmicroorganisms which produce alcohol at titers above their tolerancelevels.

Alcohol-producing microorganisms are known in the art. For example,fermentative oxidation of methane by methanotrophic bacteria (forexample, Methylosinus trichosporium) produces methanol, contactingmethanol (a C₁ alkyl alcohol) with a carboxylic acid and a catalystcapable of esterifying the carboxylic acid with methanol forms amethanol ester of the carboxylic acid. The wild-type yeast strainCEN.PK113-7D (CBS 8340, the Centraal Buro voor Schimmelculture; vanDijken J P, et al., 2000, An interlaboratory comparison of physiologicaland genetic properties of four Saccharomyces cerevisiae strains. EnzymeMicrob. Technol. 26:706-714) can produce ethanol; contacting ethanolwith a carboxylic acid and a catalyst capable of esterifying thecarboxylic acid with the ethanol forms ethyl ester.

Recombinant microorganisms which produce alcohol are also known in theart (for example, Ohta et al., 1991, Appl. Environ. Microbiol.57:893-900; Underwood et al., 2002, Appl. Environ. Microbiol.68:1071-1081; Shen and Liao, 2008, Metab. Eng. 10:312-320; Hahnai etal., 2007,Appl. Environ. Microbiol. 73:7814-7818; U.S. Pat. No.5,514,583, U.S. Pat. No. 5,712,133; PCT Application Pub. No.WO1995028476; Feldmann et al., 1992, Appl. Microbiol. Biotechnol. 38:354-361; Zhang et al., 1995, Science 267:240-243; 20070031918 A1; U.S.Pat. No. 7,223,575, U.S. Pat. No. 7,741,119; US 20090203099 A1; USApplication Pub. No. 2009/0246846 A1; and PCT Application Pub. No.WO2010/075241, which are herein incorporated by reference).

Suitable recombinant microorganisms capable of producing butanol areknown in the art, and certain suitable microorganisms capable ofproducing butanol are described herein. Recombinant microorganisms toproduce butanol via a biosynthetic pathway can include a member of thegenera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia,Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus,Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus,Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces,Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, Issatchenkia, orSaccharomyces. In one embodiment, recombinant microorganisms can beselected from the group consisting of Escherichia coli, Lactobacillusplantarum, and Saccharomyces cerevisiae. In one embodiment, therecombinant microorganism is a yeast. In one embodiment, the recombinantmicroorganism is crabtree-positive yeast selected from Saccharomyces,Zygosaccharomyces, Schizosaccharomyces, Dekkera, Torulopsis,Brettanomyces, and some species of Candida. Species of crabtree-positiveyeast include, but are not limited to, Saccharomyces cerevisiae,Saccharomyces kluyveri, Schizosaccharomyces pombe, Saccharomycesbayanus, Saccharomyces mikitae, Saccharomyces paradoxus,Zygosaccharomyces rouxii, and Candida glabrata. In some embodiments, thehost cell is Saccharomyces cerevisiae. S. cerevisiae yeast are known inthe art and are available from a variety of sources, including, but notlimited to, American Type Culture Collection (Rockville, Md.),Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre,LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts,Martrex, and Lallemand. S. cerevisiae yeast include, but are not limitedto, BY4741, CEN.PK 113-7D, Ethanol Red® yeast, Ferm Pro™ yeast,Bio-Ferm® XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, GertStrand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMax™Green yeast, FerMax™ Gold yeast, Thermosacc® yeast, BG-1, PE-2, CAT-1,CBS7959, CBS7960, and CBS7961.

Additionally, recombinant microbial production hosts comprising a1-butanol biosynthetic pathway (U.S. Patent Application Publication No.US20080182308A1, herein incorporated by reference), a 2-butanolbiosynthetic pathway (U.S. Patent Publication Nos. US 20070259410A1,herein incorporated by reference and US 20070292927, herein incorporatedby reference), and an isobutanol biosynthetic pathway (U.S. PatentPublication No. US 20070092957, herein incorporated by reference) havebeen described.

The production of butanol utilizing fermentation with a microorganism,as well as microorganisms which produce butanol, is disclosed, forexample, in U.S. Pub. No. 2009/0305370, herein incorporated byreference. In some embodiments, microorganisms comprise a butanolbiosynthetic pathway. In embodiments, at least one, at least two, atleast three, or at least four polypeptides catalyzing substrate toproduct conversions of a pathway are encoded by heterologouspolynucleotides in the microorganism. In embodiments, all polypeptidescatalyzing substrate to product conversions of a pathway are encoded byheterologous polynucleotides in the microorganism. In some embodiments,the microorganism comprises a reduction or elimination of pyruvatedecarboxylase activity. Microorganisms substantially free of pyruvatedecarboxylase activity are described in US Application Publication No.20090305363, herein incorporated by reference. Microorganismssubstantially free of an enzyme having NAD-dependentglycerol-3-phosphate dehydrogenase activity such as GPD2 are alsodescribed therein.

Butanol Biosynthetic Pathways

Certain suitable isobutanol biosynthetic pathways are disclosed in U.S.Patent Application Publication No. US 20070092957, which is incorporatedby reference herein. A diagram of the disclosed isobutanol biosyntheticpathways is provided in FIG. 2. As described in U.S. Patent ApplicationPublication No. US 20070092957 A1, which is incorporated by referenceherein, steps in an example isobutanol biosynthetic pathway includeconversion of:

-   -   pyruvate to acetolactate (see FIG. 2, pathway step a therein),        as catalyzed for example by acetolactate synthase,    -   acetolactate to 2,3-dihydroxyisovalerate (see FIG. 2, pathway        step b therein) as catalyzed for example by KAR1;    -   2,3-dihydroxyisovalerate to 2-ketoisovalerate (see FIG. 2,        pathway step c therein) as catalyzed for example by acetohydroxy        acid dehydratase, also called dihydroxy-acid dehydratase (DHAD);    -   2-ketoisovalerate to isobutyraldehyde (see FIG. 2, pathway step        d therein) as catalyzed for example by branched-chain 2-keto        acid decarboxylase; and    -   isobutyraldehyde to isobutanol (see FIG. 2, pathway step e        therein) as catalyzed for example by branched-chain alcohol        dehydrogenase.

The substrate to product conversions for steps f, g, h, i, j, and k ofalternative pathways are described in U.S. Patent ApplicationPublication No. US 2007/0092957 A1, which is incorporated by referenceherein.

Genes and polypeptides that can be used for the substrate to productconversions described above as well as those for additional isobutanolpathways, are described in U.S. Patent Appl. Pub. No. 20070092957,incorporated by reference herein. US Appl. Pub. Nos. 20070092957 and20100081154, describe dihydroxyacid dehydratase (DHAD) enzymes,including a DHAD from Streptococcus mutans (SEQ ID NO: 262) and a DHADfrom Lactococcus lactis (SEQ ID NO: 263). U.S. Patent Appl. Publ. No.2009/0269823 and 2011/0269199 A1, incorporated by reference herein,describe alcohol dehydrogenases, including an alcohol dehydrogenase fromAchromobacter xylosoxidans (SEQ ID NO: 258) and an alcohol dehydrogenasefrom Beijerinkia indica (SEQ ID NO: 259). Ketol-acid reductoisomerase(KAR1) enzymes are described in U.S. Patent Appl. Pub. Nos. 20080261230A1, 20090163376, 20100197519, and PCT Appl. Pub. No. WO/2011/041415, allincorporated by reference herein. Keto-acid decarboxylases include thosefrom Lactococcus lactis (SEQ ID NO: 260) and Listeria grayi (SEQ ID NO:261)

Additionally described in U.S. Pat. Nos. 7,851,188 and 7,993,889, whichis incorporated by reference herein, are construction of chimeric genesand genetic engineering of bacteria and yeast for isobutanol productionusing the disclosed biosynthetic pathways.

In another embodiment, the isobutanol biosynthetic pathway comprises thefollowing substrate to product conversions:

-   -   pyruvate to acetolactate, which may be catalyzed, for example,        by acetolactate synthase;    -   acetolactate to 2,3-dihydroxyisovalerate, which may be        catalyzed, for example, by ketol-acid reductoisomerase;    -   2,3-dihydroxyisovalerate to a-ketoisovalerate, which may be        catalyzed, for example, by dihydroxyacid dehydratase;    -   α-ketoisovalerate to valine, which may be catalyzed, for        example, by transaminase or valine dehydrogenase;    -   valine to isobutylamine, which may be catalyzed, for example, by        valine decarboxylase;    -   isobutylamine to isobutyraldehyde, which may be catalyzed by,        for example, omega transaminase; and,    -   isobutyraldehyde to isobutanol, which may be catalyzed, for        example, by a branched-chain alcohol dehydrogenase.

In another embodiment, the isobutanol biosynthetic pathway comprises thefollowing substrate to product conversions:

-   -   pyruvate to acetolactate, which may be catalyzed, for example,        by acetolactate synthase;    -   acetolactate to 2,3-dihydroxyisovalerate, which may be        catalyzed, for example, by acetohydroxy acid reductoisomerase;    -   2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be        catalyzed, for example, by acetohydroxy acid dehydratase;    -   α-ketoisovalerate to isobutyryl-CoA, which may be catalyzed, for        example, by branched-chain keto acid dehydrogenase;    -   isobutyryl-CoA to isobutyraldehyde, which may be catalyzed, for        example, by acetylating aldehyde dehydrogenase; and,    -   isobutyraldehyde to isobutanol, which may be catalyzed, for        example, by a branched-chain alcohol dehydrogenase.

In another embodiment, the isobutanol biosynthetic pathway comprises thesubstrate to product conversions shown as steps k, g, and e in FIG. 2.

Biosynthetic pathways for the production of 1-butanol that may be usedinclude those described in U.S. Appl. Pub. No. 2008/0182308, which isincorporated herein by reference. In one embodiment, the 1-butanolbiosynthetic pathway comprises the following substrate to productconversions:

-   -   a) acetyl-CoA to acetoacetyl-CoA, which may be catalyzed, for        example, by acetyl-CoA acetyl transferase;    -   b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, which may be        catalyzed, for example, by 3-hydroxybutyryl-CoA dehydrogenase;    -   c) 3-hydroxybutyryl-CoA to crotonyl-CoA, which may be catalyzed,        for example, by crotonase;    -   d) crotonyl-CoA to butyryl-CoA, which may be catalyzed, for        example, by butyryl-CoA dehydrogenase;    -   e) butyryl-CoA to butyraldehyde, which may be catalyzed, for        example, by butyraldehyde dehydrogenase; and,    -   f) butyraldehyde to 1-butanol, which may be catalyzed, for        example, by butanol dehydrogenase.

Biosynthetic pathways for the production of 2-butanol that may be usedinclude those described in U.S. Appl. Pub. No. 2007/0259410 and U.S.Appl. Pub. No. 2009/0155870, which are incorporated herein by reference.In one embodiment, the 2-butanol biosynthetic pathway comprises thefollowing substrate to product conversions:

-   -   a) pyruvate to alpha-acetolactate, which may be catalyzed, for        example, by acetolactate synthase;    -   b) alpha-acetolactate to acetoin, which may be catalyzed, for        example, by acetolactate decarboxylase;    -   c) acetoin to 3-amino-2-butanol, which may be catalyzed, for        example, acetonin aminase;    -   d) 3-amino-2-butanol to 3-amino-2-butanol phosphate, which may        be catalyzed, for example, by aminobutanol kinase;    -   e) 3-amino-2-butanol phosphate to 2-butanone, which may be        catalyzed, for example, by aminobutanol phosphate phosphorylase;        and,    -   f) 2-butanone to 2-butanol, which may be catalyzed, for example,        by butanol dehydrogenase.

In another embodiment, the 2-butanol biosynthetic pathway comprises thefollowing substrate to product conversions:

-   -   a) pyruvate to alpha-acetolactate, which may be catalyzed, for        example, by acetolactate synthase;    -   b) alpha-acetolactate to acetoin, which may be catalyzed, for        example, by acetolactate decarboxylase;    -   c) acetoin to 2,3-butanediol, which may be catalyzed, for        example, by butanediol dehydrogenase;    -   d) 2,3-butanediol to 2-butanone, which may be catalyzed, for        example, by dial dehydratase; and,    -   e) 2-butanone to 2-butanol, which may be catalyzed, for example,        by butanol dehydrogenase.

Methods for In Situ Product Removal

The improved microorganisms and processes described herein may be usedin conjunction with other in situ product removal processes, such aswith those described in PCT Appn. Pub No. WO2011/159998, incorporated byreference herein. FIG. 1 illustrates an example process flow diagram forproduction of product alcohol such as ethanol or butanol according to anembodiment of the present invention. As shown, a feedstock 12 can beintroduced to an inlet in a liquefaction vessel 10 and liquefied toproduce a feedstock slurry 16. Feedstock 12 contains hydrolysablepolysaccharides that supplies a fermentable carbon substrate (e.g.,fermentable sugar such as glucose), and can be a biomass such as, butnot limited to rye, wheat, cane or corn, or can otherwise be derivedfrom a biomass. In some embodiments, feedstock 12 can be one or morecomponents of a fractionated biomass, and in other embodiments,feedstock 12 can be a milled, unfractionated biomass. In someembodiments, feedstock 12 can be corn, such as dry milled,unfractionated corn kernels, and the undissolved particles can includegerm, fiber, and gluten. The undissolved solids are non-fermentableportions of feedstock 12. For purposes of the discussion herein withreference to the embodiments shown in the Figures, feedstock 12 willoften be described as constituting milled, unfractionated corn, in whichthe undissolved solids have not been separated therefrom. However, itshould be understood that the exemplary methods and systems describedherein can be modified for different feedstocks whether fractionated ornot, as apparent to one of skill in the art.

The process of liquefying feedstock 12 involves hydrolysis ofpolysaccharides in feedstock 12 into sugars, including for example,dextrins and oligosaccharides. Any known liquefying processes, as wellas the corresponding liquefaction vessel, normally utilized by theindustry can be used including, but not limited to, the acid process,the acid-enzyme process, or the enzyme process. Such processes can beused alone or in combination. In some embodiments, the enzyme processcan be utilized and an appropriate enzyme 14, for example alpha-amylase,is introduced to an inlet in liquefaction vessel 10. Water can also beintroduced to liquefaction vessel 10. In embodiments, a saccharificationenzyme, for example glucoamylase, may also be introduced to liquefactionvessel 10.

Feedstock slurry 16 produced from liquefying feedstock 12 comprisesfermentable carbon substrate (e.g. sugar), and, optionally, depending onthe feedstock, triglycerides in the form of oil and undissolved solidsderived from the feedstock. Feedstock slurry 16 can be discharged froman outlet of liquefaction vessel 10. In some embodiments, feedstock 12is corn or corn kernels and therefore feedstock slurry 16 is a corn mashslurry. In some embodiments, feedstock 12 is a lignocellulosic feedstockand therefore feedstock slurry 16 may be a lignocellulosic hydrolysate.In some embodiments, undissolved solids are removed from feedstockslurry 16 prior to introduction into the fermentation vessel.

Feedstock slurry 16 is introduced into a fermentation vessel 30 alongwith a microorganism comprising a polynucleotide encoding a polypeptidehaving lipase activity provided in accordance with the present invention32. Fermentation vessel 30 is configured to ferment slurry 16 to producealcohol. In particular, microorganism 32 contacts the fermentable carbonsubstrate in slurry 16 to produce product alcohol. The slurry caninclude a fermentable carbon source, for example, in the form ofoligosaccharides, and water.

In some embodiments, slurry 16 is subjected to a saccharificationprocess in order to break the complex sugars (e.g., oligosaccharides) inslurry 16 into monosaccharides that can be readily metabolized bymicroorganism 32. Any known saccharification process, normally utilizedby the industry can be used including, but not limited to, the acidprocess, the acid-enzyme process, or the enzyme process. In someembodiments, simultaneous saccharification and fermentation (SSF) canoccur inside fermentation vessel 30, as shown in FIG. 1. In someembodiments, an enzyme 38, such as glucoamylase, can be introduced to aninlet in fermentation vessel 30 in order to breakdown the starch oroligosaccharides to glucose capable of being metabolized bymicroorganism 32.

Carboxylic acid 28 and/or native oil containing triglycerides 26 areintroduced into fermentation vessel 30, along with an optional catalyst42. Optional catalyst 42 can be introduced before, after, orcontemporaneously with enzyme 38. Thus, in some embodiments, addition ofenzyme 38 and optional catalyst 42 can be stepwise (e.g, catalyst 42,then enzyme 38, or vice versa), or substantially simultaneous (i.e, atexactly the same time as in the time it takes for a person or a machineto perform the addition in one stroke, or one enzyme/catalystimmediately following the other catalyst/enzyme as in the time it takesfor a person or a machine to perform the addition in two strokes).Optional catalyst 42 is capable of esterifying the product alcohol withcarboxylic acid 28 to form an alcohol ester and in embodiments is apurified lipase. For example, in the case of butanol production,optional catalyst 42 is capable of esterifying butanol with carboxylicacid 28 to form a butanol ester. It is believed that catalyst 42 isoptional for use in the methods described herein because the recombinantmicroorganism will express and display or secrete into the fermentationmedium a lipase to catalyze the esterification. However, it may bedesirable to add purified lipase (optional catalyst 42) and the methodsand microorganisms provided herein allow for a reduction in the amountof optional catalyst 42 to be added.

In the instance that native oil containing triglycerides 26 is suppliedto fermentation vessel 30, at least a portion of the acyl glycerides inoil 26 can be hydrolyzed to carboxylic acid 28 by contacting oil 26 witha polypeptide having lipase activity such as secreted or displayed bythe microorganisms provided herein and/or optional catalyst 42. In someembodiments, the resulting acid/oil composition includes monoglyceridesand/or diglycerides from the partial hydrolysis of the acyl glyceridesin the oil. In some embodiments the resulting acid/oil compositionincludes glycerol, a by-product of acyl glyceride hydrolysis.

In addition, depending on the feedstock, the acyl glycerides in the oilderived from feedstock 12 and present in slurry 16 can also behydrolyzed to carboxylic acid 28. In some embodiments, the concentrationof carboxylic acids in the broth is sufficient to form a two-phasefermentation mixture comprising an organic phase and an aqueous phase.

Carboxylic acid 28 can be any carboxylic acid capable of esterifyingwith a product alcohol, such as butanol or ethanol, to produce analcohol ester of the carboxylic acid. For example, in some embodiments,carboxylic acid 28 can be free fatty acid, and in some embodiments thecarboxylic acid or free fatty acid have a chain length of 4 to 28carbons, 4 to 22 carbons in other embodiments, 8 to 22 carbons in otherembodiments, 10 to 28 carbons in other embodiments, 10 to 22 carbons inother embodiments, 12 to 22 carbons in other embodiments, 4 to 18carbons in other embodiments, 12 to 22 carbons in other embodiments, and12 to 18 carbons in still other embodiments, and 16 to 22 carbons instill other embodiments. In some embodiments, carboxylic acid 28 is oneor more of the following fatty acids: azaleic, capric, caprylic, castor,coconut (i.e., as a naturally-occurring combination of fatty acids,including lauric, myrisitic, plamitic, caprylic, capric, stearic,caproic, arachidic, oleic, and linoleic, for example), isostearic,lauric, linseed, myristic, oleic, palm oil, palmitic, palm kernel,pelargonic, ricinoleic, sebacic, soya, stearic acid, tall oil, tallow,and #12 hydroxy stearic. In some embodiments, carboxylic acid 28 is oneor more of diacids, e.g., azelaic acid and sebacic acid. In someembodiments, carboxylic acid 28 is one or more saturated, primarycarboxylic acids with defined branching of the carbon chain, where saidcarboxylic acid or mixtures thereof are prepared by the oxidation of2-alkyl-1-alkanols well known as Guerbet alcohols, where the carboxylicacids have a total number of carbons of from 12 to 22.

Thus, in some embodiments, carboxylic acid 28 can be a mixture of two ormore different fatty acids. In some embodiments, carboxylic acid 28comprises free fatty acid derived from hydrolysis of acyl glycerides byany method known in the art, including chemical or enzymatic hydrolysis.In some embodiments as noted above, carboxylic acid 28 can be derivedfrom native oil 26 by enzymatic hydrolysis of the oil glycerides usingan enzyme as catalyst 42. In some embodiments, the fatty acids ormixtures thereof comprise unsaturated fatty acids. The presence ofunsaturated fatty acids decreases the melting point, providingadvantages for handling. Of the unsaturated fatty acids, those which aremonounsaturated, i.e. possessing a single carbon-carbon double bond, mayprovide advantages with respect to melting point without sacrificingsuitable thermal and oxidative stability for process considerations.

In some embodiments, native oil 26 can be tallow, corn, canola,capric/caprylic triglycerides, castor, coconut, cottonseed, fish,jojoba, lard, linseed, neetsfoot, oiticica, palm, peanut, rapeseed,rice, safflower, soya, sunflower, tung, jatropha, pumpkin, palm, grapeseed and vegetable oil blends (or oils that can be purified into higherconcentrations of different chain length and levels of unsaturation(i.e., 18:1)). In some embodiments, native oil 26 is a mixture of two ormore native oils, such as a mixture of palm and soybean oils, forexample. In some embodiments, native oil 26 is a plant-derived oil. Insome embodiments, the plant-derived oil can be, though not necessarily,derived from biomass that can be used in a fermentation process. Thebiomass can be the same or different source from which feedstock 12 isobtained. Thus, for example, in some embodiments, oil 26 can be derivedfrom corn, whereas feedstock 12 can be cane. For example, in someembodiments, oil 26 can be derived from corn, and the biomass source offeedstock 12 is also corn. Any possible combination of different biomasssources for oil 26 versus feedstock 12 can be used, as should beapparent to one of skill in the art. In some embodiments, oil 26 isderived from the biomass used in the fermentation process. Thus, in someembodiments oil 26 is derived directly from feedstock 12. For example,when feedstock 12 is corn, then oil 26 is the feedstock's constituentcorn oil and may be introduced into fermentation vessel 30 along withslurry 16.

In fermentation vessel 30, alcohol produced by microorganism 32 isesterified with carboxylic acid 28 by the polypeptide having lipaseactivity secreted by the microorganism (and optionally catalyst 42) toform alcohol esters. For example, in the case of butanol production,butanol produced by microorganism 32 is esterified with carboxylic acid28 to form butanol esters. In situ product removal (ISPR) can beutilized to remove the alcohol esters from the fermentation broth.Utilizing a recombinant microorganism which expresses and secretes ordisplays a polypeptide having lipase activity to form esters inconjunction with ISPR can improve the performance of the fermentation.While not wishing to be bound by theory, it is believed that lipaseactivity in the fermentation medium and esterification of the productalcohol during a fermentation may improve the ability of themicroorganism to produce the product alcohol which is particularlydesirable for product alcohols that are toxic to the production hostcells. Thus, provided herein are methods of improving tolerance of amicroorganism to a product alcohol by engineering the microorganism toproduce and secrete a polypeptide having lipase activity.

In embodiments, using the microorganism to produce a lipase to formesters in conjunction with ISPR (such as, for example, liquid-liquidextraction) can increase the effective titer by at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, or at least about 100% as compared to the effectivetiter in an analogous fermentation using ISPR without the microorganismproducing a lipase. Similarly, in embodiments, using the microorganismto produce a lipase to form esters in conjunction with ISPR (such as,for example, liquid-liquid extraction) can increase the effective rateby at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, or at least about 100% ascompared to the effective rate in an analogous fermentation using ISPRwithout the microorganism producing a lipase. In embodiments, theeffective yield is increased by at least about 10%, at least about 20%,at least about 30%, at least about 40%, or at least about 50%. In someembodiments, the resulting fermentation broth after alcoholesterification can comprise free (i.e., unesterified) alcohol, and insome embodiments, the concentration of free alcohol in the fermentationbroth after alcohol esterification is not greater than 1, 3, 6, 10, 15,20, 25, 30 25, 40, 45, 50, 55, or 60 g/L when the product alcohol isbutanol, or, when the product alcohol is ethanol, the concentration offree alcohol in the fermentation broth after alcohol esterification isnot greater than 15, 20, 25, 30 25, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100 g/L. In some embodiments, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, or at least about 90% ofthe effective titer of alcohol is converted to alcohol ester.

In some embodiments, the fermentation broth is contacted duringfermentation with an extractant to form a two-phase mixture comprisingan aqueous phase and an organic phase. Such liquid-liquid extraction canbe performed according to the processes described in U.S. Pub. No.2009/0305370, the disclosure of which is hereby incorporated in itsentirety. U.S. Patent Appl. Pub. No. 2009/0305370 describes methods forproducing and recovering butanol from a fermentation broth usingliquid-liquid extraction, the methods comprising the step of contactingthe fermentation broth with a water immiscible extractant to form atwo-phase mixture comprising an aqueous phase and an organic phase.Typically, the extractant can be an organic extractant selected from thegroup consisting of saturated, mono-unsaturated, poly-unsaturated (andmixtures thereof) C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids,esters of C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, C₁₂ to C₂₂fatty amides, and mixtures thereof. The extractant may also be anorganic extractant selected from the group consisting of saturated,mono-unsaturated, poly-unsaturated (and mixtures thereof) C₄ to C₂₂fatty alcohols, C₄ to C₂₈ fatty acids, esters of C₄ to C₂₈ fatty acids,C₄ to C₂₂ fatty aldehydes, and mixtures thereof. Examples of suitableextractants include an extractant comprising at least one solventselected from the group consisting of oleyl alcohol, behenyl alcohol,cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, oleicacid, lauric acid, myristic acid, stearic acid, methyl myristate, methyloleate, lauric aldehyde, 1-nonanol, 1-decanol, 1-undecanol, 2-undecanol,1-nonanal, 2-butyloctanol, 2-butyl-octanoic acid and mixtures thereof.In embodiments, the extractant comprises oleyl alcohol. In embodiments,the extractant comprises a branched chain saturated alcohol, forexample, 2-butyloctanol, commercially available as ISOFAL® 12 (Sasol,Houston, Tex.) or Jarcol 1-12 (Jarchem Industries, Inc., Newark, N.J.).In embodiments, the extractant comprises a branched chain carboxylicacid, for example, 2-butyl-octanoic acid, 2-hexyl-decanoic acid, or2-decyl-tetradecanoic acid, commercially available as ISOCARB® 12,ISOCARB® 16, and ISOCARB® 24, respectively (Sasol, Houston, Tex.). Foruse with the processes described herein, the extractant(s) for ISPR aretypically non-alcohol extractants, so as to avoid consuming carboxylicacid 28 in fermentation vessel 30 by catalytic esterification ofcarboxylic acid 28 with an alcohol extractant, whereby less carboxylicacid would be available for esterification with the product alcohol. Forexample, if oleyl alcohol is used as an ISPR extractant, then oleylalcohol esters of the carboxylic acid may be produced in fermentationvessel due to the presence of lipase activity.

With reference to the embodiment of FIG. 1, the carboxylic acid 28 canalso serve as an ISPR extractant 28 or a component thereof. As earliernoted, carboxylic acid 28 can be supplied, and/or formed in situ in thecase when native oil 26 is supplied to fermentation vessel 30, and/orformed in situ in the case when feedstock 16 includes triglycerides inthe form of oil that can be hydrolyzed. In some embodiments, ISPRextractant 28 includes free fatty acids. In some embodiments, ISPRextractant 28 includes corn oil fatty acids (COFA). In some embodiments,oil 26 is corn oil, whereby ISPR extractant 28 is COFA. ISPR extractant(carboxylic acid) 28 contacts the fermentation broth and forms atwo-phase mixture comprising an aqueous phase 34 and an organic phase.The product alcohol ester formed in the fermentation brothpreferentially partitions into the organic phase to form anester-containing organic phase 36. Any free product alcohol in thefermentation broth also preferentially partitions into theester-containing organic phase. The biphasic mixture can be removed fromfermentation vessel 30 as stream 39 and introduced into a vessel 35, inwhich the ester-containing organic phase 36 is separated from aqueousphase 34. Separation of biphasic mixture 39 into ester-containingorganic phase 36 and aqueous phase 34 can be achieved using any methodsknown in the art, including but not limited to, siphoning, aspiration,decantation, centrifugation, using a gravity settler, membrane-assistedphase splitting, and the like. All or part of aqueous phase 34 can berecycled into fermentation vessel 30 as fermentation medium (as shown),or otherwise discarded and replaced with fresh medium, or treated forthe removal of any remaining product alcohol and then recycled tofermentation vessel 30.

With reference to FIG. 1, ester-containing organic phase 36 isintroduced into vessel 50 in which the alcohol esters are reacted withone or more substances 52 to recover product alcohol 54. Product alcohol54 can be recovered using any method known in the art and/or describedin PCT Appn. Pub. No. WO2011/159998, incorporated by reference, forobtaining an alcohol from an alcohol ester.

EXAMPLES

As used herein, the meaning of abbreviations used was as follows: “L”means liter(s), “mL” means milliliter(s), “μL” means microliter(s).

General Methods GC Analysis of Reaction Products in the Aqueous andExtractant Phase

Samples (ca. 5.0 g) were removed from a stirred reaction mixture orfermentation broth containing corn oil fatty acids (COFA) as extractant,and centrifuged to separate aqueous phase and extractant phase. A sampleof the resulting aqueous phase or extractant phase (ca. 0.50 g, actualweight recorded) was dissolved in 4.50 mL of a solution of 5.5556 mg/mLof pentadecanoic acid methyl ester (C15:0 FAME, external standard) inisopropanol. The resulting solution was centrifuged to remove anysuspended solids, then ca. 1.25 mL of the resulting supernatant wasadded to a 2.0 mL Agilent GC sample vial and the vial capped with a PTFEsepta. Samples were analyzed for isobutanol or fatty acid butyl esterson an Agilent 6890 GC with a 7683B injector and autosampler. The columnwas an Agilent DB-FFAP column (30 m×0.25 mm ID, 0.25 μm film). Thecarrier gas was helium at a flow rate of 1.8 mL/min measured at 80° C.with constant head pressure; injector split was 20:1 at 250° C.; oventemperature was 80° C. for 2.0 minutes, 80° C. to 250° C. at 10° C./min,then 250° C. for 20 minutes. Flame ionization detection was used at 250°C. The following GC standards (Nu-Chek Prep; Elysian, Minn.) were usedto confirm the identity of fatty acid isobutyl ester products: iso-butylpalmitate, iso-butyl stearate, iso-butyl oleate, iso-butyl linoleate,iso-butyl linolenate, iso-butyl arachidate.

Strain Constructions

TABLE 7 Genotypes of strains used in Examples Strain Genotype PNY827MATa/MATα PNY908 MATa MAL2-8c SUC2 PNY931 MATa ura3Δ::loxP his3Δ pdc6Δpdc1Δ::P[PDC1]- DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxPfra2Δ::P[TEF1(M6)]-LIP|Tlan-CYC1tadh1Δ::UAS(PGK1)P[FBA1]-kivD_Ll(y)-ADH1t PNY932 MATa ura3Δ::loxP his3Δpdc6Δ pdc1Δ::P[PDC1]- DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxPfra2Δ::CYC1t-LIP|Tlan-P[TEF1(M6)]adh1Δ::UAS(PGK1)P[FBA1]-kivD_Ll(y)-ADH1t PNY934 Isogenic with PNY 931,transformed with pBP915 and pYZ090ΔalsS PNY935 Isogenic with PNY 932,transformed with pBP915 and pYZ090ΔalsS PNY937 Isogenic with PNY2211,transformed with pBP915 and pYZ090ΔalsS PNY1020 MATα ura3Δ::loxP his3ΔpTVAN2 PNY1022 MATa ura3Δ::loxP his3Δ pdc6Δ pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxPfra2Δ::P[PDC1]-ADH|adh_HI-ADH1t adh1Δ::UAS(PGK1)P[FBA1]-kivD_Lg(y)-ADH1typrcΔ15Δ::P[PDC5]-ADH|adh_HI-ADH1t gpd2Δ::P[TEF1(M4)]-CdLip(y)-CYC1tpBP2092 PNY1023 MATa ura3Δ::loxP his3Δ pdc6Δ pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxPfra2Δ::P[PDC1]-ADH|adh_HI-ADH1t adh1Δ::UAS(PGK1)P[FBA1]-kivD_Lg(y)-ADH1typrcΔ15Δ::P[PDC5]-ADH|adh_HI-ADH1t gpd2Δ::P[TEF1(M6)]-CdLip(y)-CYC1tpBP2092 PNY1024 MATa ura3Δ::loxP his3Δ pdc6Δ pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxPfra2Δ::P[PDC1]-ADH|adh_HI-ADH1t adh1Δ::UAS(PGK1)P[FBA1]-kivD_Lg(y)-ADH1typrcΔ15Δ::P[PDC5]-ADH|adh_HI-ADH1t gpd2Δ::P[TEF1(M6)]-CaLip(y)-CYC1tpBP2092 PNY1052 MATa ura3Δ::loxP his3Δ pTVAN31 PNY1053 MATa ura3Δ::loxPhis3Δ pTVAN32 PNY1054 MATa ura3Δ::loxP his3Δ pTVAN33 PNY1055 MATaura3Δ::loxP his3Δ pTVAN9 PNY1056 MATa ura3Δ::loxP his3Δ pTVAN4 PNY1057MATa ura3Δ::loxP his3Δ pTVAN10 PNY1500 MATa ura3Δ::loxP his3Δ PNY1556MATa ura3Δ::loxP his3Δ pdc6Δ pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxPfra2Δ::P[PDC1]-ADH|adh_HI-ADH1t adh1Δ::UAS(PGK1)P[FBA1]-kivD_Lg(y)-ADH1typrcΔ15Δ::P[PDC5]-ADH|adh_HI-ADH1t PNY2211 MATa ura3Δ::loxP his3Δ pdc6Δpdc1Δ::P[PDC1]- DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP fra2Δadh1Δ::UAS(PGK1)P[FBA1]-kivD_Ll(y)-ADH1t PNY2242 MATa ura3Δ::loxP his3Δpdc6Δ pdc1Δ::P[PDC1]- DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxPfra2Δ::P[PDC1]-ADH|adh_HI-ADH1t adh1Δ::UAS(PGK1)P[FBA1]-kivD_Ll(y)-ADH1typrcΔ15Δ::P[PDC5]-ADH|adh_HI-ADH1t ymr226cΔ ald6Δ::loxP

TABLE 8 Feature information for constructs used in Examples Strand (W =“Watson”; Feature Position C = “Crick”) Feature information for SEQ IDNO: 183 Amp^(R) 1629-2486 C HIS3 4532-5191 W pTEF1(M6)  1-400 C Tlanlipase ORF 6433-7308 C CYC1 Terminator 6175-6424 C Feature informationfor SEQ ID NO: 184 Amp^(R) 1629-2486 C HIS3 4532-5191 W pTEF1(M6)  1-400C Tlan lipase ORF 6433-7308 C CYC1 Terminator 6175-6424 C N55A mutation7144-7146 C Feature information for SEQ ID NO: 185 Amp^(R) 4000-4860 CFragment A 431-931 W Fragment B  956-1455 W 5′ URA3 1464-1713 W URA31714-2517 W 3′ URA3 2518-2667 W Fragment C 2676-2788 W Featureinformation for SEQ ID NO: 186 Amp^(R) 3059-3919 C Fragment A 4550-5050W pTEF1(M6) 5067-5466 W Tlan lipase ORF 5476-6350 W CYC1 Terminator6360-6609 W Fragment B  15-514 W 5′ URA3 523-772 W URA3  773-1576 W 3′URA3 1577-1726 W Fragment C 1735-1847 W Feature information for SEQ IDNO: 187 Amp^(R) 3059-3919 C Fragment A 4550-5050 W CYC1 Terminator5067-5316 C Tlan lipase ORF 5326-6200 C pTEF1(M6) 6210-6609 C Fragment B 15-514 W 5′ URA3 523-772 W URA3  773-1576 W 3′ URA3 1577-1726 WFragment C 1735-1847 W Feature information for SEQ ID NO: 188 Amp^(R)4610-5470 C Fragment A 6101-6601 W pTEF1(M6)  7-406 W Tlan lipase N55AORF  416-1290 W CYC1 Terminator 1300-1549 W Fragment B 1566-2065 W 5′URA3 2074-2323 W URA3 2324-3127 W 3′ URA3 3128-3277 W Fragment C3286-3398 W

Construction of PNY1500

The strain BP857 (“PNY1500”) was derived from CEN.PK 113-7D (CBS 8340;Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre,Netherlands) and contains deletions of the following genes: URA3, HIS3.

URA3 Deletion

To delete the endogenous URA3 coding region, a ura3::loxP-kanMX-loxPcassette was PCR-amplified from pLA54 template DNA (SEQ ID NO: 25).pLA54 contains the K. lactis TEF1 promoter and kanMX marker, and isflanked by loxP sites to allow recombination with Cre recombinase andremoval of the marker. PCR was done using Phusion DNA polymerase (NewEngland BioLabs; Ipswich, Mass.) and primers BK505 and BK506 (SEQ IDNOs:26 and 27). The URA3 portion of each primer was derived from the 5′region upstream of the URA3 promoter and 3′ region downstream of thecoding region such that integration of the loxP-kanMX-loxP markerresulted in replacement of the URA3 coding region. The PCR product wastransformed into CEN.PK 113-7D using standard genetic techniques(Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selectedon YPD containing G418 (100 μg/ml) at 30° C. Transformants were screenedto verify correct integration by PCR using primers LA468 and LA492 (SEQID NOs:28 and 29) and designated CEN.PK 113-7D Δura3::kanMX.

HIS3 Deletion

The four fragments for the PCR cassette for the scarless HIS3 deletionwere amplified using Phusion High Fidelity PCR Master Mix (New EnglandBioLabs) and CEN.PK 113-7D genomic DNA as template, prepared with aGentra Puregene Yeast/Bact kit (Qiagen; Valencia, Calif.). HIS3 FragmentA was amplified with primer oBP452 (SEQ ID NO: 30) and primer oBP453(SEQ ID NO: 31), containing a 5′ tail with homology to the 5′ end ofHIS3 Fragment B. HIS3 Fragment B was amplified with primer oBP454 (SEQID NO: 32), containing a 5′ tail with homology to the 3′ end of HIS3Fragment A, and primer oBP455 (SEQ ID NO: 33), containing a 5′ tail withhomology to the 5′ end of HIS3 Fragment U. HIS3 Fragment U was amplifiedwith primer oBP456 (SEQ ID NO: 34), containing a 5′ tail with homologyto the 3′ end of HIS3 Fragment B, and primer oBP457 (SEQ ID NO: 35),containing a 5′ tail with homology to the 5′ end of HIS3 Fragment C.HIS3 Fragment C was amplified with primer oBP458 (SEQ ID NO: 36),containing a 5′ tail with homology to the 3′ end of HIS3 Fragment U, andprimer oBP459 (SEQ ID NO: 37). PCR products were purified with a PCRPurification kit (Qiagen). HIS3 Fragment AB was created by overlappingPCR by mixing HIS3 Fragment A and HIS3 Fragment B and amplifying withprimers oBP452 (SEQ ID NO: 30) and oBP455 (SEQ ID NO: 33). HIS3 FragmentUC was created by overlapping PCR by mixing HIS3 Fragment U and HIS3Fragment C and amplifying with primers oBP456 (SEQ ID NO: 34) and oBP459(SEQ ID NO: 37). The resulting PCR products were purified on an agarosegel followed by a Gel Extraction kit (Qiagen). The HIS3 ABUC cassettewas created by overlapping PCR by mixing HIS3 Fragment AB and HIS3Fragment UC and amplifying with primers oBP452 (SEQ ID NO: 30) andoBP459 (SEQ ID NO: 37). The PCR product was purified with a PCRPurification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::kanMX were made and transformedwith the HIS3 ABUC PCR cassette using a Frozen-EZ Yeast TransformationII kit (Zymo Research; Orange, Calif.). Transformation mixtures wereplated on synthetic complete media lacking uracil supplemented with 2%glucose at 30° C. Transformants with a his3 knockout were screened forby PCR with primers oBP460 (SEQ ID NO: 38) and oBP461 (SEQ ID NO: 39)using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit(Qiagen). A correct transformant was selected as strain CEN.PK 113-7DΔura3::kanMX Δhis3::URA3.

KanMX Marker Removal from the Δura3 Site and URA3 Marker Removal fromthe Δhis3 Site

The KanMX marker was removed by transforming CEN.PK 113-7D Δura3::kanMXΔhis3::URA3 with pRS423::PGAL1-cre (SEQ ID NO: 40) using a Frozen-EZYeast Transformation II kit (Zymo Research) and plating on syntheticcomplete medium lacking histidine and uracil supplemented with 2%glucose at 30° C. Transformants were grown in YP supplemented with 1%galactose at 30° C. for ˜6 hours to induce the Cre recombinase and KanMXmarker excision and plated onto YPD (2% glucose) plates at 30° C. forrecovery. An isolate was grown overnight in YPD and plated on syntheticcomplete medium containing 5-fluoro-orotic acid (0.1%) at 30° C. toselect for isolates that lost the URA3 marker. 5-FOA resistant isolateswere grown in and plated on YPD for removal of the pRS423::PGAL1-creplasmid. Isolates were checked for loss of the KanMX marker, URA3marker, and pRS423::PGAL1-cre plasmid by assaying growth on YPD-FG418plates, synthetic complete medium lacking uracil plates, and syntheticcomplete medium lacking histidine plates. A correct isolate that wassensitive to G418 and auxotrophic for uracil and histidine was selectedas strain CEN.PK 113-7D Δura3::loxP Δhis3 and designated as BP857. Thedeletions and marker removal were confirmed by PCR and sequencing withprimers oBP450 (SEQ ID NO: 41) and oBP451 (SEQ ID NO: 42) for Δura3 andprimers oBP460 (SEQ ID NO: 38) and oBP461 (SEQ ID NO: 39) for Δhis3using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit(Qiagen).

Construction of Strain PNY2205 PDC6 Deletion

The four fragments for the PCR cassette for the scarless PDC6 deletionwere amplified using Phusion High Fidelity PCR Master Mix (New EnglandBioLabs) and CEN.PK 113-7D genomic DNA as template, prepared with aGentra Puregene Yeast/Bact kit (Qiagen). PDC6 Fragment A was amplifiedwith primer oBP440 (SEQ ID NO: 18) and primer oBP441 (SEQ ID NO: 19),containing a 5′ tail with homology to the 5′ end of PDC6 Fragment B.PDC6 Fragment B was amplified with primer oBP442 (SEQ ID NO: 20),containing a 5′ tail with homology to the 3″ end of PDC6 Fragment A, andprimer oBP443 (SEQ ID NO: 21), containing a 5′ tail with homology to the5′ end of PDC6 Fragment U. PDC6 Fragment U was amplified with primeroBP444 (SEQ ID NO: 22), containing a 5′ tail with homology to the 3′ endof PDC6 Fragment B, and primer oBP445 (SEQ ID NO: 23), containing a 5′tail with homology to the 5′ end of PDC6 Fragment C. PDC6 Fragment C wasamplified with primer oBP446 (SEQ ID NO: 24), containing a 5′ tail withhomology to the 3′ end of PDC6 Fragment U, and primer oBP447 (SEQ ID NO:56). PCR products were purified with a PCR Purification kit (Qiagen).PDC6 Fragment AB was created by overlapping PCR by mixing PDC6 FragmentA and PDC6 Fragment B and amplifying with primers oBP440 (SEQ ID NO:18)and oBP443 (SEQ ID NO: 21). PDC6 Fragment UC was created by overlappingPCR by mixing PDC6 Fragment U and PDC6 Fragment C and amplifying withprimers oBP444 (SEQ ID NO: 22) and oBP447 (SEQ ID NO: 56. The resultingPCR products were purified on an agarose gel followed by a GelExtraction kit (Qiagen). The PDC6 ABUC cassette was created byoverlapping PCR by mixing PDC6 Fragment AB and PDC6 Fragment UC andamplifying with primers oBP440 (SEQ ID NO: 18) and oBP447 (SEQ ID NO:56). The PCR product was purified with a PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 were made andtransformed with the PDC6 ABUC PCR cassette using a Frozen-EZ YeastTransformation II kit (Zymo Research). Transformation mixtures wereplated on synthetic complete media lacking uracil supplemented with 2%glucose at 30° C. Transformants with a pdc6 knockout were screened forby PCR with primers oBP448 (SEQ ID NO: 57) and oBP449 (SEQ ID NO: 58)using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit(Qiagen). A correct transformant was selected as strain CEN.PK 113-7DΔura3::loxP Δhis3 Δpdc6::URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3 was grown overnight in YPDand plated on synthetic complete medium containing 5-fluoro-orotic acid(0.1%) at 30° C. to select for isolates that lost the URA3 marker. Thedeletion and marker removal were confirmed by PCR and sequencing withprimers oBP448 (SEQ ID NO: 57) and oBP449 (SEQ ID NO: 58) using genomicDNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The absenceof the PDC6 gene from the isolate was demonstrated by a negative PCRresult using primers specific for the coding sequence of PDC6, oBP554(SEQ ID NO: 59) and oBP555 (SEQ ID NO: 60). The correct isolate wasselected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 and designatedas BP891.

PDC1 Deletion ilvDSm Integration

The PDC1 gene was deleted and replaced with the ilvD coding region fromStreptococcus mutans ATCC #700610. The A fragment followed by the ilvDcoding region from Streptococcus mutans for the PCR cassette for thePDC1 deletion-ilvDSm integration was amplified using Phusion HighFidelity PCR Master Mix (New England BioLabs) and NYLA83 genomic DNA astemplate, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen).NYLA83 is a strain (construction described in U.S. App. Pub. NO.20110124060, incorporated herein by reference in its entirety) whichcarries the PDC1 deletion-ilvDSm integration described in U.S. PatentApplication Publication No. 2009/0305363 (herein incorporated byreference in its entirety). PDC1 Fragment A-ilvDSm was amplified withprimer oBP513 (SEQ ID NO: 61) and primer oBP515 (SEQ ID NO: 62),containing a 5′ tail with homology to the 5′ end of PDC1 Fragment B. TheB, U, and C fragments for the PCR cassette for the PDC1 deletion-ilvDSmintegration were amplified using Phusion High Fidelity PCR Master Mix(New England BioLabs) and CEN.PK 113-7D genomic DNA as template,prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). PDC1 Fragment Bwas amplified with primer oBP516 (SEQ ID NO: 63) containing a 5′ tailwith homology to the 3′ end of PDC1 Fragment A-ilvDSm, and primer oBP517(SEQ ID NO: 64), containing a 5′ tail with homology to the 5′ end ofPDC1 Fragment U. PDC1 Fragment U was amplified with primer oBP518 (SEQID NO: 65), containing a 5′ tail with homology to the 3′ end of PDC1Fragment B, and primer oBP519 (SEQ ID NO: 66), containing a 5′ tail withhomology to the 5′ end of PDC1 Fragment C. PDC1 Fragment C was amplifiedwith primer oBP520 (SEQ ID NO: 67), containing a 5′ tail with homologyto the 3′ end of PDC1 Fragment U, and primer oBP521 (SEQ ID NO: 68). PCRproducts were purified with a PCR Purification kit (Qiagen). PDC1Fragment A-ilvDSm-B was created by overlapping PCR by mixing PDC1Fragment A-ilvDSm (SEQ ID NO: 171) and PDC1 Fragment B and amplifyingwith primers oBP513 (SEQ ID NO: 61) and oBP517 (SEQ ID NO: 64). PDC1Fragment UC was created by overlapping PCR by mixing PDC1 Fragment U andPDC1 Fragment C and amplifying with primers oBP518 (SEQ ID NO: 65) andoBP521 (SEQ ID NO: 68). The resulting PCR products were purified on anagarose gel followed by a Gel Extraction kit (Qiagen). The PDC1A-ilvDSm-BUC cassette (SEQ ID NO: 172) was created by overlapping PCR bymixing PDC1 Fragment A-ilvDSm-B and PDC1 Fragment UC and amplifying withprimers oBP513 (SEQ ID NO: 61) and oBP521 (SEQ ID NO: 68). The PCRproduct was purified with a PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 were made andtransformed with the PDC1 A-ilvDSm-BUC PCR cassette using a Frozen-EZYeast Transformation II kit (Zymo Research). Transformation mixtureswere plated on synthetic complete media lacking uracil supplemented with2% glucose at 30° C. Transformants with a pdc1 knockout ilvDSmintegration were screened for by PCR with primers oBP511 (SEQ ID NO: 69)and oBP512 (SEQ ID NO: 70) using genomic DNA prepared with a GentraPuregene Yeast/Bact kit (Qiagen). The absence of the PDC1 gene from theisolate was demonstrated by a negative PCR result using primers specificfor the coding sequence of PDC1, oBP550 (SEQ ID NO: 71) and oBP551 (SEQID NO: 72). A correct transformant was selected as strain CEN.PK 113-7DΔura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3 was grownovernight in YPD and plated on synthetic complete medium containing5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lostthe URA3 marker. The deletion of PDC1, integration of ilvDSm, and markerremoval were confirmed by PCR and sequencing with primers oBP511 (SEQ IDNO: 69) and oBP512 (SEQ ID NO: 70) using genomic DNA prepared with aGentra Puregene Yeast/Bact kit (Qiagen). The correct isolate wasselected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSmand designated as BP907.

PDC5 Deletion sadB Integration

The PDC5 gene was deleted and replaced with the sadB coding region fromAchromobacter xylosoxidans (the sadB gene is described in U.S. PatentAppl. No. 2009/0269823, which is herein incorporated by reference in itsentirety). A segment of the PCR cassette for the PDC5 deletion-sadBintegration was first cloned into plasmid pUC19-URA3MCS. pUC19-URA3MCSis pUC19 (SEQ ID NO: 94) based and contains the sequence of the URA3gene from S. cerevisiae situated within a multiple cloning site (MCS).pUC19 contains the pMB1 replicon and a gene coding for beta-lactamasefor replication and selection in E. coli. In addition to the codingsequence for URA3, the sequences from upstream and downstream of thisgene were included for expression of the URA3 gene in yeast. The vectorcan be used for cloning purposes and can be used as a yeast integrationvector.

The DNA encompassing the URA3 coding region along with 250 bp upstreamand 150 bp downstream of the URA3 coding region from Saccharomycescerevisiae CEN.PK 113-7D genomic DNA was amplified with primers oBP438(SEQ ID NO: 89), containing BamHI, AscI, PmeI, and FseI restrictionsites, and oBP439 (SEQ ID NO: 90), containing XbaI, PacI, and NotIrestriction sites, using Phusion High-Fidelity PCR Master Mix (NewEngland BioLabs). Genomic DNA was prepared using a Gentra PuregeneYeast/Bact kit (Qiagen). The PCR product and pUC19 were ligated with T4DNA ligase after digestion with BamHI and XbaI to create vectorpUC19-URA3MCS. The vector was confirmed by PCR and sequencing withprimers oBP264 (SEQ ID NO: 91) and oBP265 (SEQ ID NO: 92).

The coding sequence of sadB and PDC5 Fragment B were cloned intopUC19-URA3MCS to create the sadB-BU portion of the PDC5 A-sadB-BUC PCRcassette. The coding sequence of sadB was amplified using pLH468-sadB(SEQ ID NO: 93) as template with primer oBP530 (SEQ ID NO: 73),containing an AscI restriction site, and primer oBP531 (SEQ ID NO: 74),containing a 5′ tail with homology to the 5′ end of PDC5 Fragment B.PDC5 Fragment B was amplified with primer oBP532 (SEQ ID NO: 75),containing a 5′ tail with homology to the 3′ end of sadB, and primeroBP533 (SEQ ID NO: 76), containing a PmeI restriction site. PCR productswere purified with a PCR Purification kit (Qiagen). sadB-PDC5 Fragment Bwas created by overlapping PCR by mixing the sadB and PDC5 Fragment BPCR products and amplifying with primers oBP530 (SEQ ID NO: 73) andoBP533 (SEQ ID NO: 76). The resulting PCR product was digested with AscIand PmeI and ligated with T4 DNA ligase into the corresponding sites ofpUC19-URA3MCS after digestion with the appropriate enzymes. Theresulting plasmid was used as a template for amplification ofsadB-Fragment B-Fragment U using primers oBP536 (SEQ ID NO: 77) andoBP546 (SEQ ID NO: 78), containing a 5′ tail with homology to the 5′ endof PDC5 Fragment C. PDC5 Fragment C was amplified with primer oBP547(SEQ ID NO: 79) containing a 5′ tail with homology to the 3′ end of PDC5sadB-Fragment B-Fragment U, and primer oBP539 (SEQ ID NO: 80). PCRproducts were purified with a PCR Purification kit (Qiagen). PDC5sadB-Fragment B-Fragment U-Fragment C was created by overlapping PCR bymixing PDC5 sadB-Fragment B-Fragment U and PDC5 Fragment C andamplifying with primers oBP536 (SEQ ID NO: 77) and oBP539 (SEQ ID NO:80). The resulting PCR product was purified on an agarose gel followedby a Gel Extraction kit (Qiagen). The PDC5 A-sadB-BUC cassette (SEQ IDNO: 173) was created by amplifying PDC5 sadB-Fragment B-FragmentU-Fragment C with primers oBP542 (SEQ ID NO: 81), containing a 5′ tailwith homology to the 50 nucleotides immediately upstream of the nativePDC5 coding sequence, and oBP539 (SEQ ID NO: 80). The PCR product waspurified with a PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSmwere made and transformed with the PDC5 A-sadB-BUC PCR cassette using aFrozen-EZ Yeast Transformation II kit (Zymo Research). Transformationmixtures were plated on synthetic complete media lacking uracilsupplemented with 1% ethanol (no glucose) at 30° C. Transformants with apdc5 knockout sadB integration were screened for by PCR with primersoBP540 (SEQ ID NO: 82) and oBP541 (SEQ ID NO: 83) using genomic DNAprepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The absence ofthe PDC5 gene from the isolate was demonstrated by a negative PCR resultusing primers specific for the coding sequence of PDC5, oBP552 (SEQ IDNO: 84) and oBP553 (SEQ ID NO: 85). A correct transformant was selectedas strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSmΔpdc5::sadB-URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3 wasgrown overnight in YPE (1% ethanol) and plated on synthetic completemedium supplemented with ethanol (no glucose) and containing5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lostthe URA3 marker. The deletion of PDC5, integration of sad B, and markerremoval were confirmed by PCR with primers oBP540 (SEQ ID NO: 82) andoBP541 (SEQ ID NO: 83) using genomic DNA prepared with a Gentra PuregeneYeast/Bact kit (Qiagen). The correct isolate was selected as strainCEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB anddesignated as BP913.

GPD2 Deletion

To delete the endogenous GPD2 coding region, a gpd2::loxP-URA3-loxPcassette (SEQ ID NO: 174) was PCR-amplified using loxP-URA3-loxP PCR astemplate DNA. loxP-URA3-loxP (SEQ ID NO: 170) contains the URA3 markerfrom pRS426 flanked by loxP recombinase sites. PCR was done usingPhusion DNA polymerase and primers LA512 (SEQ ID NO: 95) and LA513 (SEQID NO: 96). The GPD2 portion of each primer was derived from the 5′region upstream of the GPD2 coding region and 3′ region downstream ofthe coding region such that integration of the loxP-URA3-loxP markerresulted in replacement of the GPD2 coding region. The PCR product wastransformed into BP913 and transformants were selected on syntheticcomplete media lacking uracil supplemented with 1% ethanol (no glucose).Transformants were screened to verify correct integration by PCR usingprimers oBP582 (SEQ ID NO: 86) and AA270 (SEQ ID NO: 87).

The URA3 marker was recycled by transformation with pRS423::PGAL1-creand plating on synthetic complete media lacking histidine supplementedwith 1% ethanol at 30° C. Transformants were streaked on syntheticcomplete medium supplemented with 1% ethanol and containing5-fluoro-orotic acid (0.1%) and incubated at 30° C. to select forisolates that had lost the URA3 marker. 5-FOA resistant isolates weregrown in YPE (1% ethanol) for removal of the pRS423::PGAL1-cre plasmid.The deletion and marker removal were confirmed by PCR with primersoBP582 (SEQ ID NO: 86) and oBP591 (SEQ ID NO: 88). The correct isolatewas selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6Δpdc1::ilvDSm Δpdc5::sadB Agpd2::loxP and designated as BP1064(PNY1503).

FRA2 Deletion

The FRA2 deletion was designed to delete 250 nucleotides from the 3′ endof the coding sequence, leaving the first 113 nucleotides of the FRA2coding sequence intact. An in-frame stop codon was present 7 nucleotidesdownstream of the deletion. The four fragments for the PCR cassette forthe scarless FRA2 deletion were amplified using Phusion High FidelityPCR Master Mix (New England BioLabs) and CEN.PK 113-7D genomic DNA astemplate, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). FRA2Fragment A was amplified with primer oBP594 (SEQ ID NO: 99) and primeroBP595 (SEQ ID NO: 102), containing a 5′ tail with homology to the 5′end of FRA2 Fragment B. FRA2 Fragment B was amplified with primer oBP596(SEQ ID NO: 103), containing a 5′ tail with homology to the 3′ end ofFRA2 Fragment A, and primer oBP597 (SEQ ID NO: 104), containing a 5′tail with homology to the 5′ end of FRA2 Fragment U. FRA2 Fragment U wasamplified with primer oBP598 (SEQ ID NO: 105), containing a 5′ tail withhomology to the 3′ end of FRA2 Fragment B, and primer oBP599 (SEQ ID NO:106) containing a 5′ tail with homology to the 5′ end of FRA2 FragmentC. FRA2 Fragment C was amplified with primer oBP600 (SEQ ID NO: 107),containing a 5′ tail with homology to the 3′ end of FRA2 Fragment U, andprimer oBP601 (SEQ ID NO: 108). PCR products were purified with a PCRPurification kit (Qiagen). FRA2 Fragment AB was created by overlappingPCR by mixing FRA2 Fragment A and FRA2 Fragment B and amplifying withprimers oBP594 (SEQ ID NO: 99) and oBP597 (SEQ ID NO: 104). FRA2Fragment UC was created by overlapping PCR by mixing FRA2 Fragment U andFRA2 Fragment C and amplifying with primers oBP598 (SEQ ID NO: 105) andoBP601 (SEQ ID NO: 108). The resulting PCR products were purified on anagarose gel followed by a Gel Extraction kit (Qiagen). The FRA2 ABUCcassette was created by overlapping PCR by mixing FRA2 Fragment AB andFRA2 Fragment UC and amplifying with primers oBP594 (SEQ ID NO: 99) andoBP601 (SEQ ID NO: 108). The PCR product was purified with a PCRPurification kit (Qiagen).

Competent cells of PNY1503 were made and transformed with the FRA2 ABUCPCR cassette using a Frozen-EZ Yeast Transformation II kit (ZymoResearch). Transformation mixtures were plated on synthetic completemedia lacking uracil supplemented with 1% ethanol at 30° C.Transformants with a fra2 knockout were screened for by PCR with primersoBP602 (SEQ ID NO: 109) and oBP603 (SEQ ID NO: 110) using genomic DNAprepared with a Gentra Puregene Yeast/Bact kit (Qiagen). A correcttransformant was grown in YPE (yeast extract, peptone, 1% ethanol) andplated on synthetic complete medium containing 5-fluoro-orotic acid(0.1%) at 30° C. to select for isolates that lost the URA3 marker. Thedeletion and marker removal were confirmed by PCR with primers oBP602(SEQ ID NO: 109) and oBP603 (SEQ ID NO: 110) using genomic DNA preparedwith a Gentra Puregene Yeast/Bact kit (Qiagen). The absence of the FRA2gene from the isolate was demonstrated by a negative PCR result usingprimers specific for the deleted coding sequence of FRA2, oBP605 (SEQ IDNO: 111) and oBP606 (SEQ ID NO: 112). The correct isolate was selectedas strain CEN.PK 113-7D MATa ura3Δ::loxP his3Δ pdc6Δpdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t pdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5tgpd2Δ::loxP fra2Δ and designated as PNY1505 (BP1135).

ADH1 Deletion and kivD_Ll(y) Integration

The ADH1 gene was deleted and replaced with the kivD coding region fromLactococcus lactis codon optimized for expression in S. cerevisiae. Thescarless cassette for the ADH1 deletion-kivD_Ll(y) integration was firstcloned into plasmid pUC19-URA3MCS.

The kivD coding region from Lactococcus lactis codon optimized forexpression in S. cerevisiae was amplified using pLH468 (SEQ ID NO: 129)as template with primer oBP562 (SEQ ID NO: 113), containing a PmeIrestriction site, and primer oBP563 (SEQ ID NO: 114), containing a 5′tail with homology to the 5′ end of ADH1 Fragment B. ADH1 Fragment B wasamplified from genomic DNA prepared as above with primer oBP564 (SEQ IDNO: 115), containing a 5′ tail with homology to the 3′ end ofkivD_Ll(y), and primer oBP565 (SEQ ID NO: 116), containing a FseIrestriction site. PCR products were purified with a PCR Purification kit(Qiagen). kivD_Ll(y)-ADH1 Fragment B was created by overlapping PCR bymixing the kivD_Ll(y) and ADH1 Fragment B PCR products and amplifyingwith primers oBP562 (SEQ ID NO: 113) and oBP565 (SEQ ID NO: 116). Theresulting PCR product was digested with PmeI and FseI and ligated withT4 DNA ligase into the corresponding sites of pUC19-URA3MCS afterdigestion with the appropriate enzymes. ADH1 Fragment A was amplifiedfrom genomic DNA with primer oBP505 (SEQ ID NO: 117), containing a SacIrestriction site, and primer oBP506 (SEQ ID NO: 118), containing an AscIrestriction site. The ADH1 Fragment A PCR product was digested with SacIand AscI and ligated with T4 DNA ligase into the corresponding sites ofthe plasmid containing kivD_Ll(y)-ADH1 Fragment B. ADH1 Fragment C wasamplified from genomic DNA with primer oBP507 (SEQ ID NO: 119),containing a PacI restriction site, and primer oBP508 (SEQ ID NO: 120),containing a SalI restriction site. The ADH1 Fragment C PCR product wasdigested with PacI and SalI and ligated with T4 DNA ligase into thecorresponding sites of the plasmid containing ADH1 FragmentA-kivD_Ll(y)-ADH1 Fragment B. The hybrid promoter UAS(PGK1)-P_(FBA1) wasamplified from vector pRS316-UAS(PGK1)-P_(FBA1)-GUS (SEQ ID NO: 130)with primer oBP674 (SEQ ID NO: 121), containing an AscI restrictionsite, and primer oBP675 (SEQ ID NO: 122), containing a PmeI restrictionsite. The UAS(PGK1)-P_(FBA1) PCR product was digested with AscI and PmeIand ligated with T4 DNA ligase into the corresponding sites of theplasmid containing kivD_Ll(y)-ADH1 Fragments ABC. The entire integrationcassette was amplified from the resulting plasmid with primers oBP505(SEQ ID NO: 117) and oBP508 (SEQ ID NO: 120) and purified with a PCRPurification kit (Qiagen).

Competent cells of PNY1505 were made and transformed with theADH1-kivD_Ll(y) PCR cassette constructed above using a Frozen-EZ YeastTransformation II kit (Zymo Research). Transformation mixtures wereplated on synthetic complete media lacking uracil supplemented with 1%ethanol at 30° C. Transformants were grown in YPE (1% ethanol) andplated on synthetic complete medium containing 5-fluoro-orotic acid(0.1%) at 30° C. to select for isolates that lost the URA3 marker. Thedeletion of ADH1 and integration of kivD_Ll(y) were confirmed by PCRwith external primers oBP495 (SEQ ID NO: 123) and oBP496 (SEQ ID NO:124) and with kivD_Ll(y) specific primer oBP562 (SEQ ID NO: 113) andexternal primer oBP496 (SEQ ID NO: 124) using genomic DNA prepared witha Gentra Puregene Yeast/Bact kit (Qiagen). The correct isolate wasselected as strain CEN.PK 113-7D MATa ura3Δ::loxP his3E pdc6Δpdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5tgpd2Δ::loxP fra2Δ adh1Δ::UAS(PGK1)P[FBA1]-kivD_Ll(y)-ADH1t anddesignated as PNY1507 (BP1201).

Construction of Integration Vector pUC19-kan::pdc1::FBA-alsS::TRX1

The FBA-alsS-CYCt cassette was constructed by moving the 1.7 kbBbvCI/PacI fragment from pRS426::GPD::alsS::CYC (described in U.S. Pat.No. 7,851,188, which is herein incorporated by reference in itsentirety) to pRS426::FBΔ::ILV5::CYC (described in U.S. Pat. No.7,851,188, which is herein incorporated by reference in its entirety),which had been previously digested with BbvCI/PacI to release the ILV5gene. Ligation reactions were transformed into E. coli TOP10 cells andtransformants were screened by PCR using primers N98SeqF1 (SEQ ID NO:125) and N99SeqR2 (SEQ ID NO: 126). The FBA-alsS-CYCt cassette wasisolated from the vector using Bg/II and NotI for cloning intopUC19-URA3::ilvD-TRX1 at the AflII site (Klenow fragment was used tomake ends compatible for ligation). Transformants containing the alsScassette in both orientations in the vector were obtained and confirmedby PCR using primers N98SeqF4 (SEQ ID NO: 127) and N1111 (SEQ ID NO:128) for configuration “A” and N98SeqF4 (SEQ ID NO: 127) and N1110 (SEQID NO: 153) for configuration “B”. A geneticin-selectable version of the“A” configuration vector was then made by removing the URA3 gene (1.2 kbNotI/NaeI fragment) and adding a geneticin cassette. Klenow fragment wasused to make all ends compatible for ligation, and transformants werescreened by PCR to select a clone with the geneticin resistance gene inthe same orientation as the previous URA3 marker using primers BK468(SEQ ID NO: 131) and N160SeqF5 (SEQ ID NO: 154). The resulting clone wascalled pUC19-kan::pdc1::FBA-alsS::TRX1 (clone A) (SEQ ID NO: 155).

Construction of alsS Integrant Strains

The pUC19-kan::pdc1::FBA-alsS integration vector described above waslinearized with PmeI and transformed into PNY1507. PmeI cuts the vectorwithin the cloned pdc1-TRX1 intergenic region and thus leads to targetedintegration at that location (Rodney Rothstein, Methods in Enzymology,1991, volume 194, pp. 281-301). Transformants were selected on YPE plus50 μg/ml G418. Patched transformants were screened by PCR for theintegration event using primers N160SeqF5 (SEQ ID NO: 154) and oBP512(SEQ ID NO: 70). Two transformants were tested indirectly foracetolactate synthase function by evaluating the strains' ability tomake isobutanol. To do this, additional isobutanol pathway genes weresupplied on E. coli-yeast shuttle vectors (pYZ090ΔalsS and pBP915; SEQID NOs: 43 and 44, respectively). One clone was designated as PNY2205.The plasmid-free parent strain was designated PNY2204 (MATa ura3Δ::loxPhis3Δ pdc6Δpdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-pUC19-loxP-kanMX-loxP-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP fra2Δadh1Δ::UAS(PGK1)P[FBA1]-kivD_Ll(y)-ADH1t).

Construction of Strain PNY2211

PNY2211 was constructed in several steps from S. cerevisiae strainPNY1507 as described in the following paragraphs. First the strain wasmodified to contain a phosphoketolase gene. Next, an acetolactatesynthase gene (alsS) was added to the strain, using an integrationvector targeted to sequence adjacent to the phosphoketolase gene.Finally, homologous recombination was used to remove the phosphoketolasegene and integration vector sequences, resulting in a scarless insertionof alsS in the intergenic region between pdc1Δ::ilvD and the native TRX1gene of chromosome XII. The resulting genotype of PNY2211 is MATaura3Δ::loxP his3Δ pdc6Δpdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP fra2Δadh1Δ::UAS(PGK1)P[FBA1]-kivD_Ll(y)-ADH1t.

A phosphoketolase gene cassette was introduced into PNY1507 bphomologous recombination. The integration construct was generated asfollows. The plasmid pRS423::CUP1-alsS+FBA-budA (previously described inUS2009/0305363, which is herein incorporated by reference in itsentirety) was digested with NotI and XmaI to remove the 1.8 kb FBA-budAsequence, and the vector was religated after treatment with Klenowfragment. Next, the CUP1 promoter was replaced with a TEF1 promotervariant (M4 variant previously described by Nevoigt et al. Appl.Environ. Microbiol. 72: 5266-5273 (2006), which is herein incorporatedby reference in its entirety) via DNA synthesis and vector constructionservice from DNA2.0 (Menlo Park, Calif.). The resulting plasmid,pRS423::TEF(M4)-alsS was cut with StuO and MluI (removes 1.6 kb portioncontaining part of the alsS gene and CYC1 termintor), combined with the4 kb PCR product generated from pRS426::GPD-xpk1+ADH-eutD (SEQ ID NO:175) with primers N1176 (SEQ ID NO: 164) and N1177 (SEQ ID NO: 165) andan 0.8 kb PCR product DNA generated from yeast genomic DNA (ENO1promoter region) with primers N822 (SEQ ID NO: 160) and N1178 (SEQ IDNO: 166) and transformed into S. cerevisiae strain BY4741 (ATCC #201388)using gap repair cloning methodology, see Ma et al. Gene 58:201-216(1987). Transformants were obtained by plating cells on syntheticcomplete medium without histidine. Proper assembly of the expectedplasmid (pRS423::TEF1(M4)-xpk1+ENO1-eutD, SEQ ID NO: 156) was confirmedby PCR primers N821 and N1115 (SEQ ID NOs: 159 and 163, respectively)and by restriction digest (BglI). Two clones were subsequentlysequenced. The 3.1 kb TEF(M4)-xpk1 gene was isolated by digestion withSacI and NotI and cloned into the pUC19-URA3::ilvD-TRX1 vector (Clone A,cut with AflII). Cloning fragments were treated with Klenow fragment togenerate blunt ends for ligation. Ligation reactions were transformedinto E. coli Stb13 cells, selecting for ampicillin resistance. Insertionof TEF1(M4)-xpk1 was confirmed by PCR (primers N1110 (SEQ ID NO: 153)and N1114 (SEQ ID NO: 162)). The vector was linearized with AflII andtreated with Klenow fragment. The 1.8 kb KpnI-HincII geneticinresistance cassette described in WO2011159853A1 (incorporated herein byreference) was cloned by ligation after Klenow fragment treatment.Ligation reactions were transformed into E. coli Stbl3 cells, selectingfor ampicillin resistance. Insertion of the geneticin cassette wasconfirmed by PCR (primers N160SeqF5 (SEQ ID NO: 154) and BK468 (SEQ IDNO: 131)). The plasmid sequence is provided herein(pUC19-URA3::pdc1::TEF(M4)-xpk1::kan, SEQ ID NO: 157).

The resulting integration cassette (pdc1::TEF1(M4)-xpk1::KanMX::TRX1)was isolated (AscI and NaeI digestion generated a 5.3 kb band that wasgel purified) and transformed into PNY1507 using the Zymo ResearchFrozen-EZ Yeast Transformation Kit (Cat. No. T2001). Transformants wereselected by plating on YPE plus 50 μg/ml G418. Integration at theexpected locus was confirmed by PCR (primers N886 and N1214, SEQ ID NOs:161 and 167, respectively). Next, plasmid pRS423::GAL1p-Cre (SEQ ID NO:169), encoding Cre recombinase, was used to remove the loxP-flankedKanMX cassette. Proper removal of the cassette was confirmed by PCR(primers oBP512 and N160SeqF5 (SEQ ID NOs: 168 and 154, respectively)).Finally, the alsS integration plasmid described herein (SEQ ID NO: 155;pUC19-kan::pdc1::FBA-alsS::TRX1, clone A) was transformed into thisstrain using the included geneticin selection marker. Two integrantswere tested for acetolactate synthase activity by transformation withplasmids pYZ090ΔalsS (SEQ ID NO: 43) and pBP915 (SEQ ID NO: 44)transformed using Protocol #2 in Amberg, Burke and Strathern “Methods inYeast Genetics” (2005), and evaluation of growth and isobutanolproduction in glucose-containing media (methods for growth andisobutanol measurement are as follows: All strains were grown insynthetic complete medium, minus histidine and uracil containing 0.3%glucose and 0.3% ethanol as carbon sources (10 mL medium in 125 mLvented Erlenmeyer flasks (VWR Cat. No. 89095-260). After overnightincubation (30° C., 250 rpm in an Innova®40 New Brunswick ScientificShaker), cultures were diluted back to 0.2 OD (Eppendorf BioPhotometermeasurement) in synthetic complete medium containing 2% glucose and0.05% ethanol (20 ml medium in 125 mL tightly-capped Erlenmeyer flasks(VWR Cat. No. 89095-260)). After 48 hours incubation (30° C., 250 rpm inan Innova®40 New Brunswick Scientific Shaker), culture supernatants(collected using Spin-X centrifuge tube filter units, Costar Cat. No.8169) were analyzed by HPLC per methods described in U.S. Appl. Pub. No.20070092957). One of the two clones was positive and was named PNY2218.

PNY2218 was treated with Cre recombinase, and the resulting clones werescreened for loss of the xpk1 gene and pUC19 integration vectorsequences by PCR (primers N886 and N160SeqR5; SEQ ID NOs: 161 and 158,respectively). This left only the alsS gene integrated in the pdc1-TRX1intergenic region after recombination the DNA upstream of xpk1 and thehomologous DNA introduced during insertion of the integration vector (a“scarless” insertion since vector, marker gene and loxP sequences arelost). Although this recombination could have occurred at any point, thevector integration appeared to be stable even without geneticinselection, and the recombination event was only observed afterintroduction of the Cre recombinase. One clone was designated PNY2211.

Construction of Saccharomyces cerevisiae Strain PNY2242

Strain PNY2242 was constructed in several steps from PNY1507 (describedabove). First, a chimeric gene comprised of the FBA1 promoter, the alsScoding region and the CYC1 terminator was integrated into ChromosomeXII, upstream of the TRX1 gene. The sequence of the modified locus isprovided as SEQ ID No. 176. Next, two copies of a gene encoding horseliver alcohol dehydrogenase were integrated into Chromsomes VII and XVI.On Chromosome VII, a chimeric gene comprised of the PDC1 promoter, thehADH coding region and the ADH1 terminator were placed into the fra2Δlocus (the original deletion of FRA2 is described above). The sequenceof the modified locus is provided as SEQ ID No. 177. On Chromosome XVI,a chimeric gene comprised of the PDC5 promoter, the hADH coding regionand the ADH1 terminator were integrated in the region formerly occupiedby the long term repeat element YPRCdelta15. The sequence of themodified locus is provided as SEQ ID No. 178. Then the native genesYMR226c and ALD6 were deleted. Elimination of YMR226c was a scarlessdeletion of only the coding region. The sequence of the modified locusis provided as SEQ ID No. 179. The ALD6 coding region plus 700 bp ofupstream sequence were deleted using CRE-lox mediated marker removal(methodology described above), so the resulting locus contains one loxPsite. The sequence of the modified locus is provided as SEQ ID No. 180.Finally, plasmids were introduced into the strain for expression of avariant of Anaerostipes caccae KAR1 (pLH702, SEQ ID. No. 181) and DHAD(pYZ067DkivDDhADH, SEQ ID. No. 182), resulting in strain PNY2242.

Construction of PNY1528

PNY1528 (hADH Integrations in PNY2211)

Deletions/integrations were created by homologous recombination with PCRproducts containing regions of homology upstream and downstream of thetarget region and the URA3 gene for selection of transformants. The URA3gene was removed by homologous recombination to create a scarlessdeletion/integration.

The scarless deletion/integration procedure was adapted from Akada etal., Yeast, 23:399 (2006). The PCR cassette for eachdeletion/integration was made by combining four fragments, A-B-U-C, andthe gene to be integrated by cloning the individual fragments into aplasmid prior to the entire cassette being amplified by PCR for thedeletion/integration procedure. The gene to be integrated was includedin the cassette between fragments A and B. The PCR cassette contained aselectable/counter-selectable marker, URA3 (Fragment U), consisting ofthe native CEN.PK 113-7D URA3 gene, along with the promoter (250 bpupstream of the URA3 gene) and terminator (150 bp downstream of the URA3gene) regions. Fragments A and C (each approximately 100 to 500 bp long)corresponded to the sequence immediately upstream of the target region(Fragment A) and the 3′ sequence of the target region (Fragment C).Fragments A and C were used for integration of the cassette into thechromosome by homologous recombination. Fragment B (500 bp long)corresponded to the 500 bp immediately downstream of the target regionand was used for excision of the URA3 marker and Fragment C from thechromosome by homologous recombination, as a direct repeat of thesequence corresponding to Fragment B was created upon integration of thecassette into the chromosome.

YPRCΔ15 Deletion and Horse Liver adh Integration

The YPRCΔ15 locus was deleted and replaced with the horse liver adhgene, codon optimized for expression in Saccharomyces cerevisiae, alongwith the PDC5 promoter region (538 bp) from Saccharomyces cerevisiae andthe ADH1 terminator region (316 bp) from Saccharomyces cerevisiae. Thescarless cassette for the YPRCΔ15 deletion-P[PDC5]-adh_HL(y)-ADH1tintegration was first cloned into plasmid pUC19-URA3MCS.

Fragments A-B-U-C were amplified using Phusion High Fidelity PCR MasterMix (New England BioLabs; Ipswich, Mass.) and CEN.PK 113-7D genomic DNAas template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen;Valencia, Calif.). YPRCΔ15 Fragment A was amplified from genomic DNAwith primer oBP622 (SEQ ID NO: 142), containing a KpnI restriction site,and primer oBP623 (SEQ ID NO: 143), containing a 5′ tail with homologyto the 5′ end of YPRCΔ15 Fragment B. YPRCΔ15 Fragment B was amplifiedfrom genomic DNA with primer oBP624 (SEQ ID NO: 144), containing a 5′tail with homology to the 3′ end of YPRCΔ15 Fragment A, and primeroBP625 (SEQ ID NO: 189), containing a FseI restriction site. PCRproducts were purified with a PCR Purification kit (Qiagen). YPRCΔ15Fragment A—YPRCΔ15 Fragment B was created by overlapping PCR by mixingthe YPRCΔ15 Fragment A and YPRCΔ15 Fragment B PCR products andamplifying with primers oBP622 (SEQ ID NO: 142) and oBP625 (SEQ ID NO:189). The resulting PCR product was digested with KpnI and FseI andligated with T4 DNA ligase into the corresponding sites of pUC19-URA3MCSafter digestion with the appropriate enzymes. YPRCΔ15 Fragment C wasamplified from genomic DNA with primer oBP626 (SEQ ID NO: 190),containing a NotI restriction site, and primer oBP627 (SEQ ID NO: 191),containing a PacI restriction site. The YPRCΔ15 Fragment C PCR productwas digested with NotI and PacI and ligated with T4 DNA ligase into thecorresponding sites of the plasmid containing YPRCΔ15 Fragments AB. ThePDC5 promoter region was amplified from CEN.PK 113-7D genomic DNA withprimer HY21 (SEQ ID NO: 192), containing an AscI restriction site, andprimer HY24 (SEQ ID NO: 193), containing a 5′ tail with homology to the5′ end of adh_Hl(y). adh_Hl(y)-ADH1t was amplified from pBP915 (SEQ IDNO: 44) with primers HY25 (SEQ ID NO: 194), containing a 5′ tail withhomology to the 3′ end of P[PDC5], and HY4 (SEQ ID NO: 195), containinga PmeI restriction site. PCR products were purified with a PCRPurification kit (Qiagen). P[PDC5]-adh_HL(y)-ADH1t was created byoverlapping PCR by mixing the P[PDC5] and adh_HL(y)-ADH1t PCR productsand amplifying with primers HY21 (SEQ ID NO: 192) and HY4 (SEQ ID NO:195).The resulting PCR product was digested with AscI and PmeI andligated with T4 DNA ligase into the corresponding sites of the plasmidcontaining YPRCΔ15 Fragments ABC. The entire integration cassette wasamplified from the resulting plasmid with primers oBP622 (SEQ ID NO:142) and oBP627 (SEQ ID NO: 191).

Competent cells of PNY2211 were made and transformed with the YPRCΔ15deletion—P[PDC5]-adh_HL(y)-ADH1t integration cassette PCR product usinga Frozen-EZ Yeast Transformation II kit (Zymo Research; Orange, Calif.).Transformation mixtures were plated on synthetic complete media lackinguracil supplemented with 1% ethanol at 30 C. Transformants were screenedfor by PCR with primers URA3-end F (SEQ ID NO: 196) and oBP637 (SEQ IDNO: 197). Correct transformants were grown in YPE (1% ethanol) andplated on synthetic complete medium supplemented with 1% EtOH andcontaining 5-fluoro-orotic acid (0.1%) at 30 C to select for isolatesthat lost the URA3 marker. The deletion of YPRCΔ15 and integration ofP[PDC5]-adh_HL(y)-ADH1t were confirmed by PCR with external primersoBP636 (SEQ ID NO: 198) and oBP637 (SEQ ID NO: 197) using genomic DNAprepared with a YeaStar Genomic DNA kit (Zymo Research). A correctisolate of the following genotype was selected for further modification:CEN.PK 113-7D MATa ura3Δ::loxP his3Δ pdc6Apdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP fra2Δadh1Δ::UAS(PGK1)P[FBA1]-kivD_Ll(y)-ADH1 typrcΔ15Δ::P[PDC5]-ADH|adh_Hl-ADH1t.

Horse Liver adh Integration at fra2Δ

The horse liver adh gene, codon optimized for expression inSaccharomyces cerevisiae, along with the PDC1 promoter region (870 bp)from Saccharomyces cerevisiae and the ADH1 terminator region (316 bp)from Saccharomyces cerevisiae, was integrated into the site of the fra2deletion. The scarless cassette for the fra2Δ-P[PDC1]-adh_HL(y)-ADH1tintegration was first cloned into plasmid pUC19-URA3MCS.

Fragments A-B-U-C were amplified using Phusion High Fidelity PCR MasterMix (New England BioLabs; Ipswich, Mass.) and CEN.PK 113-7D genomic DNAas template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen;Valencia, Calif.). fra2Δ Fragment C was amplified from genomic DNA withprimer oBP695 (SEQ ID NO: 199), containing a NotI restriction site, andprimer oBP696 (SEQ ID NO: 137), containing a PacI restriction site. Thefra2Δ Fragment C PCR product was digested with NotI and PacI and ligatedwith T4 DNA ligase into the corresponding sites of pUC19-URA3MCS. fra2ΔFragment B was amplified from genomic DNA with primer oBP693 (SEQ ID NO:201), containing a PmeI restriction site, and primer oBP694 (SEQ ID NO:202), containing a FseI restriction site. The resulting PCR product wasdigested with PmeI and FseI and ligated with T4 DNA ligase into thecorresponding sites of the plasmid containing fra2Δ fragment C afterdigestion with the appropriate enzymes. fra2Δ Fragment A was amplifiedfrom genomic DNA with primer oBP691 (SEQ ID NO: 136), containing BamHIand AsiSI restriction sites, and primer oBP692 (SEQ ID NO: 204),containing AscI and SwaI restriction sites. The fra2Δ fragment A PCRproduct was digested with BamHI and AscI and ligated with T4 DNA ligaseinto the corresponding sites of the plasmid containing fra2Δ fragmentsBC after digestion with the appropriate enzymes. The PDC1 promoterregion was amplified from CEN.PK 113-7D genomic DNA with primer HY16(SEQ ID NO: 205), containing an AscI restriction site, and primer HY19(SEQ ID NO: 206), containing a 5′ tail with homology to the 5′ end ofadh_Hl(y). adh_Hl(y)-ADH1t was amplified from pBP915 with primers HY20(SEQ ID NO: 203), containing a 5′ tail with homology to the 3′ end ofP[PDC1], and HY4 (SEQ ID NO: 195), containing a PmeI restriction site.PCR products were purified with a PCR Purification kit (Qiagen).P[PDC1]-adh_HL(y)-ADH1t was created by overlapping PCR by mixing theP[PDC1] and adh_HL(y)-ADH1t PCR products and amplifying with primersHY16 (SEQ ID NO: 205) and HY4 (SEQ ID NO: 195).The resulting PCR productwas digested with AscI and PmeI and ligated with T4 DNA ligase into thecorresponding sites of the plasmid containing fra2Δ Fragments ABC. Theentire integration cassette was amplified from the resulting plasmidwith primers oBP691 (SEQ ID NO: 136) and oBP696 (SEQ ID NO: 137).

Competent cells of the PNY2211 variant with adh_Hl(y) integrated atYPRCΔ15 were made and transformed with the fra2Δ-P[PDC1]-adh_HL(y)-ADH1tintegration cassette PCR product using a Frozen-EZ Yeast TransformationII kit (Zymo Research). Transformation mixtures were plated on syntheticcomplete media lacking uracil supplemented with 1% ethanol at 30 C.Transformants were screened for by PCR with primers URA3-end F (SEQ IDNO: 196) and oBP731 (SEQ ID NO: 139). Correct transformants were grownin YPE (1% ethanol) and plated on synthetic complete medium supplementedwith 1% EtOH and containing 5-fluoro-orotic acid (0.1%) at 30 C toselect for isolates that lost the URA3 marker.

The integration of P[PDC1]-adh_HL(y)-ADH1t was confirmed by colony PCRwith internal primer HY31 (SEQ ID NO: 200) and external primer oBP731(SEQ ID NO: 139) and PCR with external primers oBP730 (SEQ ID NO: 138)and oBP731 (SEQ ID NO: 139) using genomic DNA prepared with a YeaStarGenomic DNA kit (Zymo Research). A correct isolate of the followinggenotype was designated PNY1528: CEN.PK 113-7D MATa ura3Δ::loxP his3Δpdc6Δ pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxPfra2Δ::P[PDC1]-ADH|adh_Hl-ADH1t adh1Δ::UAS(PGK1)P[FBA1]-kivD_Ll(y)-ADH1typrcΔ15Δ::P[PDC5]-ADH|adh_Hl-ADH1t.

Construction of PNY1556

Described here is the assembly of the constructs used to replace thechromosomal copy of kivD_Ll(y) in PNY1528 at the adh1A locus withkivD_Lg(y) or kivD_Mc(y) and construction of strain PNY1556 expressingthe kivD genes.

Deletions/integrations were created by homologous recombination with PCRproducts containing regions of homology upstream and downstream of thetarget region and the URA3 gene for selection of transformants asdescribed in the previous section.

The plasmid to integrate kivD_Lg(y) was derived from a plasmidconstructed to integrate UAS(PGK1)P[FBA1]-kivD_Ll(y) into the ADH1 locusof Saccaromyces cerevisiae. Construction of the plasmid used tointegrate UAS(PGK1)P[FBA1]-kivD_Ll(y) into the ADH1 locus is describedbelow. The plasmids were constructed in pUC19-URA3MCS.

Construction of the ADH1 Deletion/UAS(PGK1)P[FBA1]-kivD_Ll(y)Integration Plasmid

The kivD coding region from Lactococcus lactis codon optimized forexpression in Saccharomyces cerevisiae, kivD_Ll(y), was amplified usingpLH468 (SEQ ID NO: 129) as template with primer oBP562 (SEQ ID NO: 113),containing a PmeI restriction site, and primer oBP563 (SEQ ID NO: 114),containing a 5′ tail with homology to the 5′ end of ADH1 Fragment B.ADH1 Fragment B was amplified from Saccharomyces cerevisiae CEN.PK113-7D genomic DNA with primer oBP564 (SEQ ID NO: 115), containing a 5′tail with homology to the 3′ end of kivD_Ll(y), and primer oBP565 (SEQID NO: 116), containing a FseI restriction site. PCR products werepurified with a PCR Purification kit (Qiagen; Valencia, Calif.).kivD_Ll(y)-ADH1 Fragment B was created by overlapping PCR by mixing thekivD_Ll(y) and ADH1 Fragment B PCR products and amplifying with primersoBP562 (SEQ ID NO: 113) and oBP565 (SEQ ID NO 116). The resulting PCRproduct was digested with PmeI and FseI and ligated with T4 DNA ligaseinto the corresponding sites of pUC19-URA3MCS after digestion with theappropriate enzymes. ADH1 Fragment A was amplified from genomic DNA withprimer oBP505 (SEQ ID NO: 117), containing a SacI restriction site, andprimer oBP506 (SEQ ID NO: 118), containing an AscI restriction site. TheADH1 Fragment A PCR product was digested with SacI and AscI and ligatedwith T4 DNA ligase into the corresponding sites of the plasmidcontaining kivD_Ll(y)-ADH1 Fragment B. ADH1 Fragment C was amplifiedfrom genomic DNA with primer oBP507 (SEQ ID NO: 119), containing a PacIrestriction site, and primer oBP508 (SEQ ID NO: 120), containing a SalIrestriction site. The ADH1 Fragment C PCR product was digested with PacIand SalI and ligated with T4 DNA ligase into the corresponding sites ofthe plasmid containing ADH1 Fragment A-kivD_Ll(y)-ADH1 Fragment B. Thehybrid promoter UAS(PGK1)-P_(FBA1) was amplified from vectorpRS316-UAS(PGK1)-P_(FBA1)-GUS (SEQ ID NO: 130) with primer oBP674 (SEQID NO: 121), containing an AscI restriction site, and primer oBP675 (SEQID NO: 122), containing a PmeI restriction site. The UAS(PGK1)-P_(FBA1)PCR product was digested with AscI and PmeI and ligated with T4 DNAligase into the corresponding sites of the plasmid containingkivD_Ll(y)-ADH1 Fragments ABC to generate pBP1181.

kivD_Ll(y) was removed from the ADH1deletion/UAS(PGK1)P[FBA1]-kivD_Ll(y) integration plasmid pBP1181. Theplasmid was digested with PmeI and FseI and the large DNA fragment waspurified on an agarose gel followed by a gel extraction kit (Qiagen).ADH1 fragment B was amplified from pBP1181 with primer oBP821 (SEQ IDNO: 207), containing a PmeI restriction site, and primer oBP484 (SEQ IDNO: 208), containing a FseI restriction site. The ADH1 fragment B PCRproduct was digested with PmeI and FseI and ligated with T4 DNA ligaseinto the corresponding sites of the gel purified large DNA fragment. APCR fragment corresponding to the 3′ 500 bp of kivD_Ll(y) was clonedinto the resulting vector for the targeted deletion of kivD_Ll(y) inPNY1528. The fragment was amplified from pBP1181 with primers oBP822(SEQ ID NO: 209), containing a NotI restriction site, and oBP823 (SEQ IDNO: 210), containing a PacI restriction site. The fragment was digestedwith NotI and PacI and ligated with T4 DNA ligase into the correspondingsites downstream of URA3 in the above plasmid with the kivD_Ll(y)deletion after digestion with the appropriate restriction enzymes. Theresulting plasmid was designated pBP1716.

The kivD coding region from Listeria grayi codon optimized forexpression in Saccharomyces cerevisiae (SEQ ID NO: 211), kivD_Lg(y), wassynthesized by DNA2.0 (Menlo Park, Calif.). kivD_Lg(y) was amplifiedwith primers oBP828 (SEQ ID NO: 212), containing a PmeI restrictionsite, and oBP829 (SEQ ID NO: 213) containing a PmeI restriction site.The resulting PCR product was digested with PmeI and ligated with T4 DNAligase into the corresponding site in pBP1716 after digestion with theappropriate enzyme. The orientation of the cloned gene was checked byPCR with primers FBAp-F (SEQ ID NO: 97) and oBP829 (SEQ ID NO: 213). Anisolate with kivD_Lg(y) in the correct orientation was designatedpBP1719.

The kivD_Ll(y) deletion/kivD_Lg(y) integration cassette was amplifiedfrom pBP1719 with primers oBP505 (SEQ ID NO: 117) and oBP823 (SEQ ID NO:210). Competent cells of the PNY1528 were made and transformed with thePCR product using a Frozen-EZ Yeast Transformation II kit (ZymoResearch; Orange, Calif.). Transformation mixtures were plated onsynthetic complete media lacking uracil supplemented with 1% ethanol at30 C. Transformants were grown in YPE (1% ethanol) and plated onsynthetic complete medium supplemented with 1% EtOH and containing5-fluoro-orotic acid (0.1%) at 30 C to select for isolates that lost theURA3 marker. The deletion of kivD_Ll(y) and integration of kivD_Lg(y)was confirmed by PCR with primers oBP674 (SEQ ID NO: 162) and oBP830(SEQ ID NO: 214) using genomic DNA prepared with a YeaStar Genomic DNAkit (Zymo Research). A correct isolate contained kivD_Lg(y) at the samelocus and expressed from the same promoter as kivD_Ll(y) in PNY1528 andwas designated PNY1556.

Example 1 Expression of Candida deformans LIP1 Lipase in Yeast

The DNA sequence of the native LIP1 lipase from C. deformans wasobtained from GenBank (accession number AJ428393), and the open readingframe (ORF) was optimized for expression in yeast (DNA 2.0). Theresulting DNA sequence had 76% sequence identity with the wild typesequence, and encoded an identical protein.

The DNA comprising the expression-optimized ORF sequence was synthesized(DNA 2.0), and the resulting DNA molecule was cloned into a yeast-E.coli shuttle vector by gap-repair cloning (Oldenburg K R, Vo K T,Michaelis S, & Paddon C (1997) Recombination-mediated PCR-directedplasmid construction in vivo in yeast. Nucleic Acids Res 25:451-452).Briefly, the LIP1 lipase ORF was amplified using primers AK10-33_CdL5and AK10-34_CdL3 (SEQ ID NOs: 10 and 11, respectively), which include 5′regions having homology to regions in plasmid pNAK34 (SEQ ID NO: 232).The resulting PCR product was co-transformed into S. cerevisiae strainPNY1500 with pNAK34 that had been linearized with the PacI restrictionendonuclease, by lithium acetate/PEG transformation essentially asdescribed (Gietz R D & Woods R A (2006) Yeast transformation by theLiAc/SS Carrier DNA/PEG method. Methods Mol Biol 313:107-120). Thetransformation reaction was plated onto synthetic complete agar medium(Sherman F (2002) Getting started with yeast. Methods in Enzymology350:3-41) containing 2% glucose and dropout mix minus histidine(Formedium, UK, catalog number DSCK-042; SCD-His medium). Afterincubation at 30° C. for 3 d, H is colonies were picked for furtheranalysis.

LIP1 lipase-positive isolates were plated onto SC-His medium containingtributyrin and incubated at 30° C. for 3 d. The LIP1 lipase-positiveisolates had a zone of clearing around them, indicating that they weresecreting a functional lipase enzyme capable of hydrolyzing tributyrin;in contrast, a control yeast strain did not cause clearing of tributyrinin the agar medium. The plasmids from 3 isolates were recovered byplasmid rescue (Robzyk K & Kassir Y (1992) A simple and highly efficientprocedure for rescuing autonomous plasmids from yeast. Nucleic AcidsRes. 20:3790) and sequenced using M13-reverse and T7-promoter primers(SEQ ID NOs: 16 and 17, respectively) on an ABI Prism 3730xl DNAAnalyzer using BigDye Terminator Cycle Sequencing chemistry. Thesequences were a perfect match for the predicted plasmid product of thegap-repair cloning strategy (data not shown). The resulting plasmid ispNAK10 (SEQ ID NO: 45; FIG. 3).

Example 2 Expression of Thermomyces lanuginosus Lipase in Yeast

The DNA sequence of the native lipase from Thermomyces lanuginosus (Tlanlipase) was obtained from GenBank (accession number AF054513), and thesequence was optimized for expression in yeast (DNA 2.0). The resultingDNA sequence had 76% sequence identity with the wildtype sequence, andencoded an identical protein.

The DNA comprising the expression-optimized ORF sequence was synthesized(DNA 2.0), and the resulting DNA molecule was cloned into a yeast-E.coli shuttle vector by gap-repair cloning as in Example 1. Briefly, thesynthesized T. lanuginosus Tlan lipase ORF was amplified using primersAK10-42_Tl5-1 and AK10-43_Tl3 (SEQ ID NOs: 12 and 13, respectively),which include 5′ regions having homology to regions in plasmid pNAK10(SEQ ID NO: 45; FIG. 3). The resulting PCR product was co-transformedinto S. cerevisiae strain PNY1500 with pNAK10 that had been linearizedwith the SpeI restriction endonuclease, by lithium acetate/PEGtransformation. The transformation reaction was plated onto SCD-Hismedium). After incubation at 30° C. for 3 d, colonies were analyzed forplasmid containing the Tlan lipase sequence by colony PCR using primersAK10-41_Tl5-1 and AK10-42_Tl3 (SEQ ID NOs: 12 and 13).

Tlan lipase-positive isolates were plated onto SCD-His medium containingtributyrin and incubated at 30° C. for 3 d. The Tlan lipase-positiveisolates had a zone of clearing around them, indicating that they weresecreting a functional lipase activity; in contrast, a control yeaststrain did not cause clearing of tributyrin in the agar medium. Theplasmids from three isolates were recovered by plasmid rescue andsequenced using M13-reverse and T7-promoter primers (SEQ ID NOs: 16 and17). The sequences were a perfect match for the predicted plasmidproduct of the gap-repair cloning strategy (data not shown). One plasmidwas named pTVAN2 (SEQ ID NO: 100).

Example 3 Scale-Up Expression of T. lanuginosus Lipase in Yeast

One positive isolate from Example 2, PNY1020, was pre-cultured overnightin SCD-His medium, and this was used to inoculate four 500 mL culturesof SCD-His medium; two cultures were treated with the asparaginylglycosylation inhibitor tunicamycin (5 μg/mL; Sigma-Aldrich, St. LouisMo.). The flasks were incubated at 30° C. and 250 rpm in a shakingincubator. After 8 h 50 mL of YPD medium (yeast extract, 10 g/L;peptone, 20 g/L; glucose, 20 g/L) was added to each flask, and thecultures were incubated overnight. The following morning, after glucosewas exhausted, the cultures were centrifuged at 8000 rpm for 10 min at4° C. The supernatants were concentrated approximately 500-fold underpressure through a 10,000 dalton molecular weight cutoff filter. Theprotein concentration of the retentates was measured, and 20 μg ofprotein was analyzed by SDS-polyacrylamide gel electrophoresis, using a4-12% acrylamide Bis-Tris gel (Invitrogen, Carlsbad Calif.) according tothe manufacturer's instructions. The gel was stained with Coomassie BlueR-250, and destained. The tunicamycin-treated protein had a lowermolecular weight, as demonstrated by its higher mobility in the gel (notshown). The identity of the band as Tlan lipase was confirmed byamino-terminal sequencing.

The concentration of Tlan lipase protein (expressed with or withouttunicamycin treatment) in the retentates was estimated to be 25% oftotal soluble protein based on SDS-PAGE analysis, and these tworetentates containing Tlan lipase protein (expressed with or withouttunicamycin treatment) were employed as catalyst for in-vitroesterification of isobutanol with corn oil fatty acids (Example 5).

Example 4 Production of Corn Oil Fatty Acids

A 5-L round bottom flask was equipped with a mechanical stirrer,thermocouple, heating mantle, condenser and nitrogen tee and chargedwith 750 g of crude corn oil, 2112 g of water and 285 g of 50% sodiumhydroxide solution. Mixture was heated to 90° C. and held for two hours,during which time it became a thick, emulsion-like single phase. At theend of this time thin-layer chromatography indicated no remaining cornoil in the mixture. The mixture was then cooled to 74° C. and 900 g of25% sulfuric acid was added to acidify the mixture, which was thencooled to 50° C. and the aqueous layer was separated. The oil layer waswashed twice with 1500 mL of 40° C. water and then once with 1 L ofsaturated brine, and then dried over magnesium sulfate and filteredthrough Celite. Yield was 610 g of clear red oil. Titration for FreeFatty Acids via AOCS method Ca 5a-40 shows a fatty acid content of 95%expressed as oleic acid. A sample (104 mg) was silanized by reactionwith 100 uL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide in 1 mL ofdry pyridine. Gas chromatography-mass spectrometry (GCMS) analysis ofthe silanized product indicated the presence of the TMS derivatives ofthe 16:0, 18:2, 18:1, 18:0, and 20:0 carboxylic acids.

Example 5 Production of Isobutyl-COFA Esters by Reaction of Isobutanoland Corn Oil Fatty Acids Catalyzed by Secreted Lipase

Reaction mixtures containing 3.6 g isobutanol (2-methyl-1-propanol),14.7 g corn oil fatty acids (COFA) prepared from corn oil (Example 4),45.1 g of aqueous 2-(N-morpholino)ethanesulfonic acid buffer (0.20 M, pH5.4), and either 0.487 mg (10 ppm in aqueous phase; Table 7) or 0.974 mg(20 ppm in aqueous phase; Table 9) of Tlan lipase protein (expressedwith or without tunicamycin treatment; Example 3) were stirred at 30°C., and samples were withdrawn from each reaction mixture atpredetermined times, immediately centrifuged, and the aqueous andorganic layers separated and analyzed for isobutanol (iBuOH) andisobutyl-esters of corn oil fatty acids (iBuO-COFA) (Table 10). Thereactions containing Tlan lipase produced considerably more iBuO-COFAthan the control reaction; the lipase samples that were secreted fromyeast in the presence of tunicamycin produced considerably moreiBuO-COFA than that produced without the inhibitor being present.

TABLE 9 Tlan concentrations in reactions for conversion of isobutanol(iBuOH) to iso-butyl esters of corn oil fatty acids (iBuO-COFA). Tlanexpressed with Reaction (ppm) tunicamycin 1 10 no 2 20 no 3 10 yes 4 20yes 5 0 not applicable

TABLE 10 Weights of isobutanol (iBuOH) and isobutyl esters of corn oilfatty acids (iBuO-COFA) present in the aqueous fraction (AQ) and organicfraction (ORG) for reactions described in Table 9. free iBuO- totaltotal iBuOH iBuOH from COFA time iBuOH iBuOH (g) iBuO-COFA (g) reaction(h) (g) (AQ) (g) (ORG) (ORG) (g) (ORG) (ORG) 1 0.1 1.45 2.15 2.14 0.010.03 1 16 1.27 2.33 2.31 0.02 0.09 1 21 1.27 2.33 2.30 0.03 0.14 1 471.24 2.36 2.28 0.08 0.36 1 89 1.23 2.37 2.22 0.15 0.67 2 0.1 1.22 2.392.38 0.01 0.03 2 16 1.29 2.32 2.30 0.03 0.11 2 21 1.25 2.36 2.32 0.040.17 2 47 1.38 2.23 2.14 0.09 0.38 2 89 1.21 2.40 2.18 0.22 0.98 3 0.11.22 2.43 2.42 0.01 0.03 3 16 1.28 2.37 2.28 0.09 0.41 3 21 1.24 2.412.29 0.12 0.55 3 47 1.22 2.43 2.15 0.28 1.27 3 89 1.17 2.48 1.94 0.542.42 4 0.1 1.38 2.22 2.21 0.01 0.03 4 16 1.30 2.30 2.19 0.11 0.49 4 211.21 2.39 2.23 0.15 0.69 4 47 1.36 2.24 1.90 0.34 1.51 4 89 1.12 2.481.78 0.70 3.16 5 0.1 1.29 2.30 2.30 0.01 0.03 5 16 1.27 2.32 2.30 0.020.08 5 21 1.24 2.35 2.33 0.02 0.10 5 47 1.35 2.24 2.20 0.05 0.21 5 891.25 2.35 2.27 0.07 0.33

Example 6 Production of Fatty Acid Butyl Esters During Yeast Cultivation

The Tlan lipase isolate PNY1020 and the control strain PNY908 werepre-cultured in SCD-His medium, and used to inoculate flasks (withnon-vented caps) containing 25 mL of SC-His medium. The flasks wereamended with 8.25 g sterile COFA (33% w/w), isobutanol (0.50 g, addedafter 8 h of growth), and tunicamycin (Tnm, final concentration 5 μg/ml)as follows (Table 11):

TABLE 11 PNY908 PNY1020 Flask COFA iBuOH Tnm flask COFA iBuOH Tnm F1 − −− F5 − − − F2 + − − F6 + − − F3 − + − F7 − + − F4 + + − F8 + + −F9 + + + F10 + − +The flasks were incubated at 30° C. and 250 rpm, and sampled after 24 hand 96 h of incubation. Samples were analyzed for glucose, ethanol,isobutanol, and fatty acid alkyl esters in the aqueous phase by HPLC orGC, and for isobutanol and fatty acid alkyl esters in the organic phaseby GC (Tables 12 and 13). When both isobutanol and COFA were added tothe cultures, the lipase-expressing strain (flasks F8 and F9) producedmore iBuO-COFA than the control strain (flask F4). The cells treatedwith tunicamycin produced more ester than the cells without inhibitortreatment.

TABLE 12 HPLC analysis of aqueous F1-F10 samples. time glucose glycerolacetate ethanol iBuOH sample (h) (mM) (mM) (mM) (mM) (mM) F1 24 0.1 2.58.2 170.4 0.8 F2 24 0.1 3.3 7.3 179.4 1.2 F3 24 50.9 1.6 3.5 106.4 266.6F4 24 0.0 1.5 5.5 182.0 144.3 F5 24 0.1 3.9 10.5 171.1 0.5 F6 24 0.1 5.07.9 184.7 0.1 F7 24 89.9 1.1 2.4 39.0 267.2 F8 24 0.1 1.8 1.7 189.9144.6 F9 24 65.9 1.7 2.6 80.7 143.5 F10 24 10.8 16.6 5.4 141.6 0.4 F1 960.0 0.7 91.7 0.3 F2 96 0.0 3.1 7.0 179.3 0.1 F3 96 50.6 1.2 3.0 108.6247.9 F4 96 0.0 1.6 7.2 183.8 130.0 F5 96 0.0 3.3 9.2 163.9 0.1 F6 960.0 4.9 9.7 182.6 0.1 F7 96 90.3 0.9 1.5 38.6 248.6 F8 96 0.0 1.9 2.0190.7 128.1 F9 96 0.7 2.1 1.2 184.2 128.5 F10 96 0.0 17.8 2.2 153.2 0.1

TABLE 13 Weights of isobutanol (iBuOH) and isobutyl esters of corn oilfatty acids (iBuO-COFA) present in the aqueous fraction (AQ) and organicfraction (ORG) for shake flask cultures described in Table 11. totaliBuO- total iBuOH free iBuOH from COFA time iBuOH (mg) iBuOH iBuO-COFA(mg) flask (h) (mg) (AQ) (ORG) (mg) (ORG) (mg) (ORG) (ORG) F1 24 0 F1 960 F2 24 0 0 0 0.0 0.0 F2 96 0 0 0 0.0 0.0 F3 24 439 F3 96 447 F4 24 239262 257 4.7 21.1 F4 96 247 255 239 15.4 69.5 F5 24 0 F5 96 0 F6 24 0 0 00.0 0.0 F6 96 0 0 0 0.0 0.0 F7 24 446 F7 96 443 F8 24 231 271 266 5.022.5 F8 96 234 267 251 16.0 72.1 F9 24 234 267 262 4.9 22.0 F9 96 224278 261 16.6 74.9 F10 24 0 0 0 0.0 0.0 F10 96 0 0 0 0.0 0.0

Example 7 Expression of Candida antarctica Lipase B in Yeast

The DNA sequence for the Candida antarctica lipase B (CalB lipase) wasobtained from GenBank (accession number Z30645), and the sequence wasoptimized for expression in yeast (DNA 2.0, Menlo Park, Calif.). Theresulting DNA sequence had 72% sequence identity with the wildtypesequence, and encoded an identical protein.

The DNA comprising the expression-optimized CalB open reading frame(ORF) sequence was synthesized (DNA 2.0), and the resulting DNA moleculewas cloned into a yeast-E. coli shuttle vector by gap-repair cloning.Briefly, the CalB lipase ORF was amplified using primers CALBL_gap_forand CALBL_gap_rev (SEQ ID NOs: 14 and 15), which include 5′ regionshaving homology to regions in plasmid pNAK34 (SEQ ID NO: 232). Theresulting PCR product was co-transformed into S. cerevisiae strainPNY1500 with plasmids pNAK33 (SEQ ID NO: 231), pNAK34 (SEQ ID NO: 232),or pNAK35 (SEQ ID NO: 233) that had been linearized with the HpaIrestriction endonuclease, by lithium acetate/PEG transformation. Thetransformation reaction was plated onto SCD-His medium. After incubationat 30° C. for 3 days, colonies were analyzed for plasmid containing theCalB lipase sequence by colony PCR using primers CALBL_gap_for andCALBL_gap_rev (SEQ ID NOs: 14 and 15).

CalB lipase-positive isolates were plated onto SCD-His medium containingtributyrin and incubated at 30° C. for 3 days. The CalB lipase-positiveisolates had a zone of clearing around them, indicating that they weresecreting a functional lipase activity; in contrast, a control yeaststrain did not cause clearing of tributyrin in the agar medium. Theplasmids from 3 isolates were recovered by plasmid rescue and sequencedusing M13-reverse and T7-promoter primers (SEQ ID NOs: 16 and 17). Thesequences were a perfect match for the predicted plasmid product of thegap-repair cloning strategy. The resulting plasmids were named pTVAN7(TEF1(M2) promoter), pTVAN3 (TEF1(M4) promoter), and pTVAN8 (TEF1(M6)promoter) (SEQ ID NOs: 278, 101, and 240, respectively).

Example 8 Surface Display of Tlan Lipase

A domain that tethers the secreted T. lanuginosus lipase to the yeastcell surface was introduced as follows. Yeast genomic DNA (PNY1500) wasused as template in a PCR reaction with primers AK11-46 and AK11-47 (SEQID NOs: 215 and 216, respectively), which amplified the codons for theC-terminal 320 amino acids of the yeast α-agglutinin protein encoded bySAG1, and added a sequence at the 5′ end containing a glycine- andserine-rich linker region. Amplification was done with Phusion DNApolymerase (New England Biolabs) according to the manufacturer'sinstructions.

This GS-SAG1 DNA was TOPO cloned into pCR-BluntII-TOPO (InVitrogen) andtransformed into DH5α. The pGS-SAG1 plasmid (SEQ ID NO: 217) wasrecovered by mini-prep (Qiagen) and the correct sequence was confirmedby DNA sequencing. The DNA was amplified with primers Sagtgap1 andSagtgap2 (SEQ ID NOs: 218 and 219, respectively) which include regionsof homology for gap-repair cloning into lipase expression vectorspTVAN11, pTVAN12, and pTVAN13 (SEQ ID NOs: 220, 221, and 222,respectively). The purified PCR products were transformed into yeaststrain PNY1500 along with PacI-digested pTVAN11 (TEF1(M2) promoter),pTVAN12 (TEF1(M4) promoter), or pTVAN13 (TEF1(M6) promoter). Thetransformation reactions were plated to SCD-His medium; colonies thatappeared tested positive for expression of lipase activity on tributyrinplates. Plasmids were rescued from these isolates (Yeast PlasmidMiniprep Kit, Zymo Research) and transformed into E. coli DH5α andpurified. Sequence analysis showed the expected nucleotide sequence ofthe lipase-SAG1 chimera.

The lipase-expressing strains (PNY1052, PNY1053, and PNY1054) and thecontrol strain (PNY1500) were grown overnight in 50 mL SCD-His medium,in a 250 mL vented-cap flask incubated at 30° C. and 250 rpm. Thefollowing morning, 21.5 mL of the culture was transferred to a 125 mLflask (unvented cap), with addition of 1.75 mL glucose (500 g/L), 2.5 mL10×YEP (100 g/L yeast extract, 200 g/L peptone), and 0.313 mLisobutanol. A sample was taken, then 10.3 mL COFA and a sterile stir barwere added and the flasks returned to incubation. A sample (5 mL) wastaken after 24 h for HPLC and GC analysis, and 1.75 mL glucose and 0.313mL isobutanol were added. A second sample was taken after 72 h. Sampleswere analyzed as described above (Table 14). The strains expressing theSAG1-lipase chimera produced more fatty acid butyl ester (FABE) than thecontrol strain. Strain PNY1054, which had the strongest promoter drivingtranscription of the chimera, produced greater than 6-fold more FABEthan the control, whereas the strains with weaker promoters producedonly ˜30% more FABE than the control.

TABLE 14 Measured amounts of isobutanol (iBuOH) and fatty acid isobutylester (FABE) in aqueous and organic phases of shake flask cultivationsof the strain indicated. iBuOH in rxn, mg iBuOH in rxn, mg FABE in rxn,mg Strain (AQ) (ORG) (ORG) 24 h PNY1052 226 255 29 PNY1053 224 252 30PNY1054 230 248 167 PNY1500 221 245 25 72 h PNY1052 201 204 52 PNY1053197 202 52 PNY1054 206 188 264 PNY1500 195 196 39

Example 9 Cell Surface Display: Cell-Association Test

This experiment was conducted to determine whether the lipase activityexpressed by the SAG1-lipase chimera was in fact cell-associated or wassecreted into the culture broth.

The lipase-expressing strains (PNY1052, PNY1053, and PNY1054) and thecontrol strain (PNY1500) were grown for 24 h in 25 mL SCD-His medium(6.7 g/L yeast nitrogen base without amino acids, 1926 mg dropout mixminus histidine, 20 g/L glucose), in a 250 mL vented-cap flask incubatedat 30° C. and 250 rpm. Then the cells and culture broth were separatedby centrifugation; the cell pellet was washed twice and resuspended in25 mL 50 mM MES buffer pH 5.5. The spent cell-free culture medium wasamended with 1 M MES buffer pH 5.5 to 50 mM. Isobutanol (finalconcentration 20 g/L) and COFA (33% wt/wt) were added to the spentmedium and to the cell suspension. The two reactions were incubated for72 h at 30° C. and 250 rpm. Samples were analyzed by GC as describedabove (Table 15). The suspensions of lipase-expressing cells formed˜2.5-fold more FABE than the control cell suspension, after incubationfor 72 h with COFA and isobutanol. In contrast, there was no differencein FABE accumulation in the cell-free medium samples, demonstrating thatthe Sag1-lipase chimeric protein is exclusively cell-associated underthese conditions.

TABLE 15 Measured amounts of isobutanol (iBuOH) and fatty acid isobutylester (FABE) in aqueous and organic phases of shake flask cultivationsof the strain indicated. iBuOH in rxn, mg iBuOH in rxn, mg FABE in rxn,mg Strain (AQ) (ORG) (ORG) Cell-free medium PNY1052 215.1 236.3 56.2PNY1053 211.0 236.8 56.1 PNY1054 221.1 269.1 63.5 PNY1500 208.7 256.660.3 Cells suspended in MES buffer PNY1052 226.3 204.1 143.0 PNY1053224.2 209.2 151.9 PNY1054 230.3 211.6 137.8 PNY1500 221.1 225.8 55.9

Example 10 Engineering Isobutanol-Producing Yeast to Secrete T.lanuginosus Lipase

The Tlan lipase transgene was amplified from plasmid pTVAN6 (SEQ ID NO:183) with oligonucleotides AK11-24 (SEQ ID NO: 132) and AK11-25 (SEQ IDNO: 133), which include AscI sites at their 5′ ends. The PCR productswere digested with AscI and ligated into AscI-digested pBP1236 (SEQ IDNO: 185). This plasmid is used to apply the technique of Akada et al.(Akada R et al. (2006) PCR-mediated seamless gene deletion and markerrecycling in Saccharomyces cerevisiae. Yeast 23:399-405) for integrationof transgenes at the fra2Δ locus of yeast. The ligation mixture wastransformed into competent E. coli DH5α (Invitrogen, Carlsbad Calif.)and plated onto LB-ampicillin agar. Colonies from this plate were grownovernight in LB-ampicillin, and plasmid DNA was isolated using theQiaprep Spin Miniprep kit. Recombinant plasmids were identified bydigestion with AscI and agarose gel electrophoresis. DNA sequencing wasused to identify the orientation of the lipase transgenes in theconstruct. Plasmid pNAK15 (SEQ ID NO: 186) contains the wildtype lipasetransgene in the reverse direction, and pNAK16 (SEQ ID NO: 187) containsthe wildtype lipase transgene in the forward orientation.

The lipase transgenes were amplified from these plasmids along withflanking DNA that targets them for integration at fra2Δ (and whichincludes the URA3 gene as a selectable marker) using primers oBP691 (SEQID NO: 136) and oBP696 (SEQ ID NO: 137). The PCR products were purifiedand concentrated using a QIAQuick PCR Purification kit. Yeast strainPNY2211 (construction described above) was grown overnight in YPE medium(10 g/L yeast extract, 20 g/L peptone, 20 mL/l 95% ethanol) at 30° C.and 250 rpm, and transformed with the PCR products followed by platingto SCE-Ura agar medium (6.7 g/L yeast nitrogen base without amino acids(YNB; Difco 291940, BD, Franklin Lakes N.J.), 1926 mg/L dropout mix -Ura(DSCK102, Formedium, Norfolk UK), 20 mL/L 95% ethanol). Ura⁺ colonieswere plated to fresh medium, and then re-plated to FOA medium (6.7 g/LYNB, 1 g/L 5-fluoroorotic acid, 200 mg/L uracil, 20 mL/195% ethanol) toselect for isolates that had lost the URA3 selection marker.

FOA-resistant transformants were checked for correct integration of thetransgene and loss of the selection marker by colony PCR using primerpairs for each flank of the integration cassette as follows: for theconstruct with the transgene in the forward orientation (from pNAK16),primer pairs AK11-26 (SEQ ID NO: 134) and oBP730 (SEQ ID NO: 138), andAK11-27 (SEQ ID NO: 135) and oBP731 (SEQ ID NO: 139), were used; for theconstruct with the transgene in the reverse orientation (from pNAK15 andpNAK17), primer pairs AK11-27 (SEQ ID NO: 135) and oBP730 (SEQ ID NO:138), and AK11-26 (SEQ ID NO: 134) and oBP731 (SEQ ID NO: 139), wereused. Isolates that produced the correct PCR products were chosen forfurther study, and named PNY931 (reverse orientation), PNY932 (forwardorientation), and PNY933 (reverse orientation, N55A mutation).

Mutagenesis of the asparagine at residue 55 of Tlan lipase (in theN-glycosylation site) to alanine was carried out with the QuikChangeSite-Directed Mutagenesis Kit (Strategene, La Jolla Calif.) according tothe manufacturer's instructions, combining plasmid pNAK15 (SEQ ID NO:186) and the following primers:

SEQ ID NO Primer Sequence 140 TILipNAforGATGCCCCAGCAGGGACAGCTATTACATGTACTGG AAACGC 141 TILipNArevGCGTTTCCAGTACATGTAATAGCTGTCCCTGCTGG GGCATC

After amplification of the plasmid backbone with mutagenic primers usingthe thermostable polymerase provided with the kit, the DNA was digestedwith DpnI restriction endonuclease. The treated plasmids weretransformed into E. coli XL1-Blue competent cells, and recovered usingthe Qiaprep Spin Miniprep Kit (Qiagen, Valencia, Calif.). Mutated cloneswere identified by DNA sequence analysis of the mutagenized plasmids.One such plasmid was named pNAK17 (SEQ ID NO: 189).

The lipase integrant yeast strains, and their parent strain PNY2211,were transformed with plasmids pBP915 (SEQ ID NO: 44) and pYZ090ΔalsS(SEQ ID NO: 43) in order to introduce an isobutanol metabolic pathway.The strains were cultivated overnight in YPE medium, then transformedwith plasmid DNA as described above, and plated to SCE-His-Ura agarmedium (6.7 g/L YNB, 1850 mg/L dropout mix -His-Ura (DSCK162,Formedium), 20 mL/195% ethanol). Colonies were re-plated to SCE-His-Uraagar medium, and named PNY934,PNY935, and PNY936.

Example 11 Production of Isobutanol and Fatty Acid Isobutyl Esters byHeterologous Lipase Expression

Strains PNY934 and PNY935 were replated to SC-His-Ura DE agar medium(6.7 g/L YNB, 1850 mg/L dropout mix -His-Ura, 3 g/L glucose, 3 mL/195%ethanol); these cells, along with cells of a control strain PNY2242(which produces isobutanol but does not secrete heterologous lipase)were used to inoculate 3 mL pre-cultures of SC-His-Ura DE medium. Thesewere grown ˜6 h. Two mL were used to inoculate 50 mL of the same mediumin 250 mL flasks with vented caps; these were grown overnight to anoptical density (OD₆₀₀) of ˜1. The next morning, glucose, yeast extract,and peptone were added to concentrations of 35, 10, and 20 g/L,respectively, with a final volume of 75 mL. This was divided evenlyamong triplicate 125 mL shake flasks (non-vented caps, containing a stirbar), and then 10.3 mL of corn oil fatty acid (COFA) was added and theflasks were incubated at 30° C. and 250 rpm. Samples were taken at 0 h(0.6 mL, before COFA addition), and at 24 h and 72 h (5 mL each, afterthorough mixing of the aqueous and COFA phases). Samples were analyzedby HPLC and GC as previously described. At 24 h, 1.5 mL of 500 g/Lglucose was added.

The isobutanol produced in these fermentations was distributed among 3fractions: free isobutanol in the aqueous and COFA phases, and a fattyacid isobutyl ester (FABE) fraction produced by the esterification ofisobutanol with fatty acids. As shown in Table 16, the lipase-secretingstrains produced considerable amounts of FABE, whereas the controlstrain produced only a low amount, ˜12% of that produced by thelipase-secreting strains. The lipase-catalyzed esterification ofisobutanol into FABE resulted in a decrease in the aqueous isobutanolconcentration as a percent of the total amount of isobutanol in thesystem by about 10%, from 53% to 43% at 24 h, and from 45% to ˜31% at 72h.

TABLE 16 Measured amounts of isobutanol (iBuOH) and fatty acid isobutylester (FABE) in aqueous and organic phases of shake flask cultivationsof the strain indicated. Mean ± standard deviation of triplicate flasksis shown. Amounts are corrected for volume loss due to sampling. iBuOHin rxn, mg iBuOH in rxn, mg FABE in rxn, mg (AQ) (ORG) (ORG) 24 h PNY93442.2 ± 0.9   32 ± 3.4 103.3 ± 4.7  PNY935 49.7 ± 14.9 41.3 ± 15.6 106.5± 13.8 PNY2242  93 ± 0.4 78.3 ± 2.5  15.1 ± 0.1 72 h PNY934 37.9 ± 3.2 32.8 ± 1.1    283 ± 43.3 PNY935 50.4 ± 20.8  32 ± 1.2 284.4 ± 57.6PNY2242 97.6 ± 57.3 112.5 ± 31.8  34.9 ± 3.5

Example 12 Production of Isobutanol and Fatty Acid Isobutyl Esters byHeterologous Lipase Secretion Comparing Glycosylated andNon-Glycosylated Lipase

The experiment of the previous example was repeated, with the inclusionof strain PNY936, which secretes the N55A mutant of Tlan lipase. In thisexperiment, glucose was added twice, after 24 h and again after 48 h(1.5 mL of 500 g/L glucose).

The lipase-secreting strains produced more FABE than the control strain(in this instance ˜5-6-fold more). The proportion of the totalisobutanol in the aqueous fraction was decreased as a consequence ofFABE formation, by ˜10% at 24 h and by ˜15% at 72 h. The cultures inwhich lipase-secreting isobutanologens were grown produced significantlymore FABE fraction than the control. The amount of FABE produced in thefermentation with the PNY936 strain (secreting glycosylation-mutantlipase) did not differ significantly from that produced by the strainssecreting the wildtype lipase enzyme.

TABLE 17 Measured amounts of isobutanol (iBuOH) and fatty acid isobutylester (FABE) in aqueous and organic phases of shake flask cultivationsof the strain indicated. Mean ± standard deviation of triplicate flasksis shown. Amounts are corrected for volume loss due to sampling. iBuOHin rxn, mg iBuOH in rxn, mg FABE in rxn, mg (AQ) (ORG) (ORG) 24 h PNY93436.3 ± 6.5 31.3 ± 5.8 89.1 ± 6.8 PNY935 33.2 ± 2   27.5 ± 1.4 76.5 ± 5.5PNY936 34.8 ± 1   28.8 ± 0.8 74.5 ± 1.3 PNY2242 75.1 ± 4.3 63.4 ± 3    14 ± 0.1 72 h PNY934 48.3 ± 6.2   41 ± 4.6 197.6 ± 20.3 PNY935 45.2 ±3.6 39.6 ± 5.8 184.2 ± 13.4 PNY936 46.4 ± 1.8 38.7 ± 1.3 203.7 ± 24.4PNY2242 143.4 ± 20.8 135.9 ± 22.7 34.3 ± 2.1

Example 13 Engineering Isobutanol-Producing Yeast to Express C.deformans and C. antarctica Lipases

The LIP1 and CalB lipase transgenes encoding the wildtype lipases fromC. deformans and C. antarctica, respectively, were amplified fromplasmids pNAK10 (SEQ ID NO: 45), pNAK31 (SEQ ID NO: 238), and pTVAN8(SEQ ID NO: 240) with oligonucleotides AK11-24 (SEQ ID NO: 132) andAK11-25 (SEQ ID NO: 133), which include AscI sites at their 5′ ends. ThePCR products were digested with AscI and ligated into AscI-digestedpNAK36 (SEQ ID NO: 223). The ligation mixture was transformed intocompetent E. coli DH5α (Invitrogen) and plated onto LB-ampicillin agar.Colonies from this plate were grown overnight in LB-ampicillin, andplasmid DNA was isolated using the Qiaprep Spin Miniprep kit.Recombinant plasmids were identified by digestion with AscI and agarosegel electrophoresis. Plasmid pNAK38 (SEQ ID NO: 224) contains the CalBlipase under control of the TEF1(M6) promoter, pNAK37 (SEQ ID NO: 225)contains the LIP1 lipase under control of the TEF1(M4) promoter, andpNAK39 (SEQ ID NO: 226) contains the LIP1 lipase under control of theTEF1(M6) promoter.

The lipase transgenes were amplified from these plasmids along withflanking DNA that targets them for integration at gpd2Δ (and whichincludes the URA3 gene as a selectable marker) using primers oBP691 (SEQID NO: 136) and oBP696 (SEQ ID NO: 137). The PCR products were purifiedand concentrated using a QIAQuick PCR Purification kit. Yeast strainPNY1556 was grown overnight in YPE medium (10 g/l yeast extract, 20 g/lpeptone, 20 ml/195% ethanol) at 30° C. and 250 rpm, and transformed withthe PCR products followed by plating to SCE-Ura agar medium.Ura⁺colonies were plated to fresh medium, and then re-plated to FOAmedium to select for isolates that had lost the selectable marker.

FOA-resistant transformants were checked for correct integration of thetransgene and loss of the selectable marker by colony PCR using primerpairs for each flank of the integration cassette as follows: genomic DNAwas purified using the PureGene kit (Qiagen) essentially as described bythe manufacturer. This was used as template for a PCR reaction witholigos HY48 (SEQ ID NO: 227) and HY49 (SEQ ID NO: 228). Positiveintegrants were plated to FOA medium, and FOA-resistant isolates wererecovered. Isolates which had lost the URA3 marker from the gpd2Δ locuswere identified by PCR using oligos HY48 and HY49 as described above.

The lipase integrant yeast strains and the control strain, PNY1556, weretransformed with plasmid pBP2092 (SEQ ID NO: 237) in order to introducean isobutanol metabolic pathway, as follows: The strains were cultivatedovernight in YPE medium, then transformed with plasmid DNA as describedabove, and plated to SCE-His agar medium. Colonies were re-plated toSCE-Ura agar medium, and named PNY1022 (TEF1(M4)-LIP1), PNY1023(TEF1(M6)-LIP1), and PNY1024 (TEF1(M4)-CalB).

Example 14 Production of Isobutanol and Fatty Acid Isobutyl Esters byHeterologous Expression of C. deformans and C. antarctica Lipases in anIsobutanologen

Strains PNY1022, PNY1023, and PNY1024 were replated to SC-Ura DE agarmedium; these cells were used to inoculate 3 ml pre-cultures ofSC-His-Ura DE medium, which were grown ˜6 h. Two ml were used toinoculate 50 ml of the same medium in 250 ml flasks with vented caps;these were grown overnight to an optical density (OD₆₀₀) of ˜1. The nextmorning glucose, yeast extract, and peptone were added to concentrationsof 35, 10, and 20 g/l, respectively, with a final volume of 75 ml. Thiswas divided evenly among triplicate 125 ml shake flasks (non-ventedcaps, containing a stir bar), 10.3 ml of corn oil fatty acid (COFA) wasadded, and the flasks were incubated at 30° C. and 250 rpm. Samples weretaken at 0 h (before COFA addition), and at 24 h, 48 h, and 94 h, afterthorough mixing of the aqueous and COFA phases. Samples were analyzed byHPLC and GC. At 24 h, 1.5 mL of 500 g/L glucose was added (1.2 mL to thePNY1556 culture); at 48 h, 1.6 mL of glucose was added to each flask.

The isobutanol produced in these fermentations was distributed among 3phases: free isobutanol in the aqueous and COFA phases, and fatty acidisobutyl ester (FABE) produced the esterification of isobutanol withfatty acids. As shown in Table 18, the strains expressing the C.deformans lipase produced considerable amounts of FABE at both 24 and 94h. PNY1023, which has a stronger promoter driving expression of the C.deformans lipase transgene, makes approx. twice as much FABE as PNY1022.Interestingly, by 94 h PNY1023 produced significantly more totalisobutanol than the other strains.

The strain expressing the CalB lipase produced much less FABE than thestrains expressing the C. deformans enzyme, although there wassignificantly more FABE in its flasks than in the control fermentations.The control strain (with no lipase transgene) esterified only 10 mg ofisobutanol into FABE by 94 h, presumably due to endogenous lipaseactivity. The fermentations carried out by lipase-expressingisobutanologens are all marked by a significantly lower aqueousisobutanol concentration than the control fermentations. In the case ofthe fermentations with C. deformans-expressing strains (PNY1022 andPNY1023), this corresponds with a significant accumulation of FABE; thestrain expressing the C. antarctica accumulated much less ester. For theC. deformans-expressing strains, the total isobutanol production iscomparable to the no-lipase control when a weak promoter is used toexpress the lipase gene; when a strong promoter is used, the totalisobutanol production is 24% higher than the control.

TABLE 18 Measured amounts of isobutanol (iBuOH) and fatty acid isobutylester (FABE) in aqueous and organic phases of shake flask cultivationsof the strain indicated. iBuOH in rxn, mg iBuOH in rxn, mg FABE in rxn,mg Strain (AQ) (ORG) (ORG) 24 h PNY1022 126 ± 3 98 ± 2 290.7 ± 16.6PNY1023  94 ± 5 67 ± 0 534.2 ± 26.1 PNY1024 157 ± 4 133 ± 4  23.7 ± 2  PNY1556 187 ± 5 112 ± 3   7.6 ± 0.5 94 h PNY1022 211 ± 3 139 ± 3  559.8± 8.3  PNY1023  206 ± 19 126 ± 14 1182.7 ± 14.3  PNY1024  204 ± 37 143 ±31 131.1 ± 11.7 PNY1556 271 ± 4 195 ± 1  42.8 ± 0.6

Example 15 Expression of Asperqillus tubinqensis LIP3 Lipase in Yeast

The DNA encoding the Aspergillus tubingensis LIP3 lipase was synthesized(DNA 2.0) with codon usage optimized for expression in S. cerevisiae.This DNA was amplified using primers Atublip1 and AtubLip2 (SEQ ID NOs:229 and 230, respectively) with Phusion DNA polymerase (New EnglandBiolabs). The PCR product was transformed into yeast strain PNY1500along with gapped plasmids pNAK33 (TEF1(M2) promoter), pNAK34 (TEF1(M4)promoter), and pNAK35 (TEF1(M6) promoter) (SEQ ID NOs: 231, 232, and233, respectively). The transformation reactions were plated to SCD-Hismedium; colonies that appeared tested positive for expression of lipaseactivity on tributyrin plates. Plasmids were rescued from these isolates(Yeast Plasmid Miniprep Kit, Zymo Research) and transformed into E. coliDH5α and purified. Sequence analysis showed the expected nucleotidesequence of the A. tubingensis lipase transgenes. They were namedpTVAN9, pTVAN4, and pTVAN10, respectively, for the TEF1(M2), TEF1(M4),and TEF1(M6) promoter variants, respectively ((SEQ ID NOs: 234, 235, and236, respectively)).

The lipase-expressing strains PNY1055 (pTVAN9), PNY1056 (pTVAN4), andPNY1057 (pTVAN10) and the wildtype control strain (PNY827) were grownovernight in 50 mL SCD-His medium in a 250 mL vented-cap flask incubatedat 30° C. and 250 rpm. The following morning, 22 mL of the culture wastransferred to a 125 mL flask (unvented cap), with addition of 1.75 mLglucose (500 g/L), 2.5 mL 10×YEP (100 g/L yeast extract, 200 g/Lpeptone), and 0.313 mL isobutanol. A sample was taken, then 10.3 mL COFAand a sterile stir bar were added and the flasks returned to incubation.A sample (5 mL) was taken after 24 h for HPLC and GC analysis, and 1.75mL glucose and 0.313 mL isobutanol were added. A second sample was takenafter 96 h. Samples were analyzed as described above. Thelipase-expressing strains were able to esterify isobutanol into FABE; at96 h, the amount of FABE formed by these strains was more than ten-foldthe amount formed by the control strain. As shown, the greatest amountof FABE formation was achieved by the strain with anintermediate-strength promoter driving lipase transcription.

TABLE 19 Measured amounts of isobutanol (iBuOH) and fatty acid isobutylester (FABE) in aqueous and organic phases of shake flask cultivationsof the strain indicated. iBuOH in rxn, mg iBuOH in rxn, mg FABE in rxn,mg Strain (AQ) (ORG) (ORG) 24 h PNY1055 108 101 183 PNY1056 98 83 272PNY1057 103 91 209 PNY827 130 116 15 96 h PNY1055 171 159 649 PNY1056141 122 886 PNY1057 163 144 700 PNY827 237 232 52

Example 16 Genetic Abolition of the Glycosylation of Lipase Expressed inYeast

N-glycosylation sequences matching to the consensus site of asparaginylglycosylation, N-X-S/T (Drickamer K & Taylor M E (2006) Introduction toGlycobiology (2nd ed.). Oxford University Press, USA) were identified inLIP1 and CalB. LIP1 has two glycosylation sites (NIS at residue 146 andNNT at residue 167), and CalB has one (NDT at residue 99). These werealtered by site-directed mutagenesis to substitute N with A in all cases(and to create the double mutant in LIP1) as follows.

Mutagenesis was carried out with the QuikChange Site-DirectedMutagenesis Kit (Strategene, La Jolla Calif.) according to themanufacturer's instructions, combining the following plasmids andprimers:

Primer Plasmid SEQ ID SEQ ID Protein, site Primers NOs: Plasmid NO:CalB, N99 Ca_NA99_for 264 pTVAN8 240 Ca_NA99_rev 265 LIP1, N146Cd_N146A_for 266 pNAK31 238 Cd_N146A_rev 267 LIP1, N167 Cd_NA167_for 268pNAK31 238 Cd_NA167_rev 269 LIP1/N167, Cd_N146A_for 266 pTVAN26 270 N146Cd_N146A_rev 267

After amplification of the plasmid backbone with mutagenic primers usingthe thermostable polymerase provided with the kit, the DNA was digestedwith DpnI restriction endonuclease. The treated plasmids weretransformed into E. coli XL1-Blue competent cells, and recovered usingthe Qiaprep Spin Miniprep Kit (Qiagen). Mutated clones were identifiedby DNA sequence analysis of the mutagenized plasmids. The plasmids werenamed pTVAN20, pTVAN25, pTVAN26, and pTVAN27, respectively. The plasmids(and control plasmids with the wildtype lipase genes) were transformedinto the PNY1500 yeast strain.

1. A method comprising: a) providing a fermentation medium comprisingfermentable carbon substrate derived from a biomass feedstock, alcoholproduced from a fermentable carbon substrate derived from a biomassfeedstock, and an alcohol-producing yeast microorganism wherein thealcohol-producing microorganism comprises an engineered polynucleotideencoding a polypeptide having lipase activity and the microorganismexpresses and displays or secretes said polypeptide such that the lipaseactivity is present in the fermentation medium; b) contacting thefermentation medium with a carboxylic acid wherein the lipase activityis present in the fermentation medium in sufficient amount to convert atleast a portion of the alcohol produced by the microorganism to alcoholesters extracellularly
 2. The method of claim 1 further comprisingcontacting the fermentation medium with an extractant to form atwo-phase mixture comprising an aqueous phase and an organic phase. 3.The method of claim 2 wherein the extractant comprises the carboxylicacid.
 4. The method of claim 1 wherein the product alcohol is a C₂ to C₈alkyl alcohol.
 5. The method of claim 1 wherein the product alcohol isethanol.
 6. The method of claim 5 wherein the alcohol esters comprisefatty acid ethyl esters.
 7. The method of claim 1 wherein the productalcohol is butanol.
 8. The method of claim 7 wherein the alcohol esterscomprise fatty acid butyl esters.
 9. (canceled)
 10. (canceled)
 11. Themethod of claim 1 wherein the polypeptide having lipase activitycomprises a sequence having at least about 70% identity to any one ofSEQ ID NOs: 249, 250, 251, 252, 253 or a fragment thereof.
 12. Themethod of claim 1 wherein the polynucleotide encoding a polypeptidehaving lipase activity comprises a sequence with at least about 70%identity to a polynucleotide having SEQ ID NO: 1, 3, 5, 7, 8, 9, 46, 48,50, 52, 54, 255, 271 or
 273. 13. The method of claim 1 wherein thepolypeptide having lipase activity comprises a sequence with at leastabout 70% identity to a polypeptide having SEQ ID NO: 2, 4, 6, 256, 47,49, 51, 53, 55, 241, 242, 243, 244, 245, 246, 247, 248, 272, or 274 oran active fragment thereof.
 14. The method of claim 13 wherein thepolypeptide having lipase activity does not contain a glycosylationmotif.
 15. The method of claim 1 wherein the polypeptide having lipaseactivity is not glycosylated.
 16. The method of claim 1 wherein thecarboxylic acid comprises free fatty acids derived from corn oil, canolaoil, palm oil, linseed oil, jatropha oil, or soybean oil.
 17. Themethod, of claim 1 wherein the carboxylic acid is derived from the samebiomass feedstock as the fermentable carbon substrate.
 18. The method ofclaim 1 wherein the carboxylic acid comprises carboxylic acids havingC₁₂ to C₂₂ linear or branched aliphatic chains.
 19. The method of claim3 wherein the contacting with extractant and the contacting withcarboxylic acid occur contemporaneously.
 20. The method of claim 1wherein at least about 60% of the effective titer of alcohol produced bythe microorganism is converted to alcohol esters.
 21. The method ofclaim 1 wherein the fermentation medium further comprises triglycerides,diglycerides, monoglycerides, and phospholipids, or combinations thereofand wherein the lipase activity hydrolyzes at least a portion of thetriglycerides, diglycerides, monoglycerides, and phospholipids, orcombinations thereof to form free fatty acids.
 22. The method of claim 1wherein the effective titer or the effective rate of alcohol producedduring a fermentation is greater than that produced during afermentation by an alcohol-producing microorganism that does notcomprise a polynucleotide encoding a polypeptide having lipase activityand the microorganism expresses and secretes or displays saidpolypeptide such that the lipase activity is present in the fermentationmedium.
 23. (canceled)
 24. A recombinant host cell comprising: (a) anengineered alcohol production pathway; and (b) an engineeredpolynucleotide encoding a polypeptide having lipase activity.
 25. Therecombinant host cell of claim 24 wherein the polypeptide having lipaseactivity comprises a sequence having at least about 70% identity to SEQID NO: 2, 4, 6, 256, 47, 49, 51, 53, 55, 241, 242, 243, 244, 245, 246,247, 248, 272, or 274 or an active fragment thereof.
 26. The recombinanthost cell of claim 24 wherein the polypeptide having lipase activitycomprises a sequence having at least about 70% identity to any one ofSEQ ID NOs: 249, 250, 251, 252, 253 or a fragment thereof.
 27. Therecombinant host cell of claim 26 wherein the polypeptide having lipaseactivity does not contain a glycosylation motif.
 28. The recombinanthost cell of claim 24 wherein the polypeptide having lipase activity isnot glycosylated.
 29. The recombinant host cell of claim 24 wherein theengineered polynucleotide encoding a polypeptide having lipase activitycomprises a sequence having at least about 70% identity to SEQ ID NO: 1,3, 5, 7, 8, 9, 46, 48, 50, 52, 54, 255, 271 or
 273. 30. A recombinanthost cell comprising: (a) an alcohol production pathway; and (b) anengineered polynucleotide encoding a polypeptide having lipase activitywherein the polypeptide having lipase activity comprises a sequencehaving at least about 70% identity to SEQ ID NO: 2, 4, 6, 256, 47, 49,51, 53, 55, 241, 242, 243, 244, 245, 246, 247, 248, 272, or 274 or anactive fragment thereof.
 31. The recombinant host cell of claim 30wherein the polypeptide having lipase activity further comprises asequence having at least about 70% identity to any one of SEQ ID NOs:249, 250, 251, 252, 253 or a fragment thereof.
 32. The recombinant hostcell of claim 24 wherein the alcohol production pathway is a butanolproduction pathway.
 33. The recombinant host cell of claim 32 whereinthe butanol production pathway is an isobutanol production pathway 34.The recombinant host cell of claim 24 wherein the host cell furthercomprises reduced or eliminated pyruvate decarboxylase activity.
 35. Amethod of increasing tolerance of an alcohol-producing microorganism tothe produced alcohol, the method comprising: (a) engineering amicroorganism to express and secrete or display a polypeptide havinglipase activity; (b) contacting the engineered microorganism with i.triglycerides, diglycerides, monoglycerides, phospholipids, free fattyacids, or a mixture thereof; and ii. a carbon substrate; underconditions whereby the microorganism produces an alcohol.
 36. The methodof claim 35 wherein the engineered microorganism is contacted withtriglycerides, diglycerides, monoglycerides, and phospholipids, orcombinations thereof and wherein the secreted or displayed lipaseconverts at least a portion of the triglycerides, diglycerides,monoglycerides, and phospholipids, or combinations thereof into freefatty acids.
 37. The method of claim 35 wherein the lipase catalyzes theformation of alcohol esters.
 38. The method of claim 35 wherein themicroorganism produces alcohol at an effective titer greater than thatproduced by a microorganism that has not been engineered to express andsecrete a polypeptide with lipase activity.
 39. The method of claim 35wherein the alcohol biosynthetic pathway is an engineered alcoholbiosynthetic pathway.
 40. The method of claim 39 wherein the engineeredalcohol biosynthetic pathway is an isobutanol biosynthetic pathway. 41.(canceled)
 42. A method of producing butyl esters during a fermentationcomprising (a) providing a fermentation medium comprising a carbonsubstrate and triglycerides, diglycerides, monoglycerides, andphospholipids, or a mixture thereof; and (b) contacting the fermentationmedium with an alcohol-producing microorganism comprising a butanolbiosynthetic pathway wherein said microorganism further comprises anengineered polynucleotide encoding a polypeptide having lipase activityand which expresses and secretes or displays the polypeptide such thatthe lipase activity is present in the fermentation medium.
 43. Themethod of claim 42 wherein the fermentation medium further comprises oneor more carboxylic acids.
 44. The method of claim 42 wherein the carbonsubstrate is derived from biomass.
 45. (canceled)
 46. (canceled)
 47. Afermentation medium comprising an alcohol-producing microorganismcomprising a butanol biosynthetic pathway and further comprising anengineered polynucleotide encoding a polypeptide having lipase activitywhich is expressed and secreted or displayed, butyl esters, and butanol.48. An animal feed product comprising a microorganism of claim 24.